Exposure to hypomethylating 5-aza-2'-deoxycytidine (decitabine) causes rapid, severe DNA damage, telomere elongation and mitotic dysfunction in human WIL2-NS cells.

BACKGROUND: 5-aza-2'-deoxycytidine (5azadC, decitabine) is a DNA hypomethylating agent used in the treatment of myelodysplastic syndromes. Due to cytotoxic side effects dose optimization is essential. The aim of this study was to define and quantify the effects of 5azadC on biomarkers of chromosomal stability, and telomere length, in human lymphoblastoid cell line, WIL2-NS, at clinically relevant dosages. METHODS: Human WIL2-NS cells were maintained in complete medium containing 0, 0.2 or 1.0 µM 5azadC for four days, and analysed daily for telomere length (flow cytometry), chromosomal stability (cytokinesis-block micronucleus cytome (CBMN-cyt) assay), and global methylation (%5me-C). RESULTS: DNA methylation decreased significantly in 1.0 µM 5azadC, relative to control (p < 0.0001). Exposure to 1.0 µM 5azadC resulted in 1.7-fold increase in telomere length (p < 0.0001), in parallel with rapid increase in biomarkers of DNA damage; (micronuclei (MN, 6-fold increase), nucleoplasmic bridges (NPB, a 12-fold increase), and nuclear buds (NBud, a 13-fold increase) (all p < 0.0001). Fused nuclei (FUS), indicative of mitotic dysfunction, showed a 5- and 13-fold increase in the 0.2 µM and 1.0 µM conditions, respectively (p = 0.001) after 4 days. CONCLUSIONS: These data show that (i) clinically relevant concentrations of 5azadC are highly genotoxic; (ii) hypomethylation was associated with increased TL and DNA damage; and (iii) longer TL was associated with chromosomal instability. These findings suggest that lower doses of 5azdC may be effective as a hypomethylating agent, while potentially reducing DNA damage and risk for secondary disease.

Hepatic Overexpression of CD36 Improves Glycogen Homeostasis and Attenuates High-Fat Diet-Induced Hepatic Steatosis and Insulin Resistance.

The common complications in obesity and type 2 diabetes include hepatic steatosis and disruption of glucose-glycogen homeostasis, leading to hyperglycemia. Fatty acid translocase (FAT/CD36), whose expression is inducible in obesity, is known for its function in fatty acid uptake. Previous work by us and others suggested that CD36 plays an important role in hepatic lipid homeostasis, but the results have been conflicting and the mechanisms were not well understood. In this study, by using CD36-overexpressing transgenic (CD36Tg) mice, we uncovered a surprising function of CD36 in regulating glycogen homeostasis. Overexpression of CD36 promoted glycogen synthesis, and as a result, CD36Tg mice were protected from fasting hypoglycemia. When challenged with a high-fat diet (HFD), CD36Tg mice showed unexpected attenuation of hepatic steatosis, increased very low-density lipoprotein (VLDL) secretion, and improved glucose tolerance and insulin sensitivity. The HFD-fed CD36Tg mice also showed decreased levels of proinflammatory hepatic prostaglandins and 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictive and proinflammatory arachidonic acid metabolite. We propose that CD36 functions as a protective metabolic sensor in the liver under lipid overload and metabolic stress. CD36 may be explored as a valuable therapeutic target for the management of metabolic syndrome.

Folate deficiency induces dysfunctional long and short telomeres; both states are associated with hypomethylation and DNA damage in human WIL2-NS cells.

The essential role of dietary micronutrients for genome stability is well documented, yet the effect of folate deficiency or excess on telomeres is not known. Accordingly, human WIL2-NS cells were maintained in medium containing 30, 300, or 3,000 nmol/L folic acid (FA) for 42 days to test the hypothesis that chronic folate deficiency would cause telomere shortening and dysfunction. After 14 days, telomere length (TL) in FA-deficient (30 nmol/L) cultures was 26% longer than that of 3,000 nmol/L FA cultures; however, this was followed by rapid telomere attrition over the subsequent 28 days (P trend, P < 0.0001); both long and short telomere status was positively correlated with biomarkers of chromosome instability (P ≤ 0.003) and mitotic dysfunction (P = 0.01), measured by the cytokinesis-block micronucleus cytome (CBMN-cyt) assay. The early increase in TL was associated with FA-deficiency-induced global DNA hypomethylation (P = 0.05), with an effect size similar to that induced by the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. Quantitative PCR analysis indicated a negative association between FA concentration and uracil incorporation into telomeric DNA (r = -0.47, P = 0.1), suggesting a possible plausible mechanism for uracil as a cause of folate deficiency-induced telomere dysfunction or deletion. Peptide nucleic acid-FISH (PNA-FISH) analysis showed that FA deficiency resulted in 60% of micronuclei containing acentric terminal fragments, an observation consistent with the 3-fold increase in terminal deletions (P = 0.0001). Together, these results demonstrate the impact of folate deficiency on biomarkers of telomere maintenance and integrity, and provide evidence that dysfunctional long telomeres may be as important as critically short telomeres as a cause of chromosomal instability.

Folate deficiency is associated with the formation of complex nuclear anomalies in the cytokinesis-block micronucleus cytome assay.

Chromosomal instability (CIN) is an important hallmark to oncogenesis and can be diagnosed morphologically by the presence of nuclear anomalies such as micronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBuds). We have identified additional nuclear anomalies formed under folate-deficient conditions, defined as "fused" nuclei (FUS), "circular" nuclei (CIR), and "horse-shoe" nuclei (HS) and investigated their suitability for inclusion as additional CIN biomarkers in the lymphocyte cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Although the morphological appearance of FUS, CIR, and HS suggested an origin from multiple NPB in the fusion region between the two nuclei, the very low frequency of dicentric chromosomes in metaphase spreads from these cultures did not support this model. Fluorescence in situ hybridization (FISH) analysis of cytokinesis-blocked binucleated (BN) cells with peptide nucleic acid probes for telomeres and centromeres (PNA-FISH) revealed a high proportion of fusion regions contained both centromeric and telomeric DNA. This suggests that folate deficiency may disrupt the process of sister chromatid separation and chromosome segregation during mitosis. It was concluded that the FUS, CIR, and HS morphologies represent promising biomarkers of CIN that are sensitive to folate deficiency, and further validation and investigation of the mechanisms responsible for their formation is warranted.

Efficacy and mechanisms of action of vitamin D in experimental polyarthritis.

Vitamin D (vit D) status has been linked to the occurrence and severity of auto-immune and inflammatory diseases. This study evaluates the effects of vit D status on adoptive transfer of adjuvant-induced arthritis (ATA). Rats maintained on diets replete or deficient in vit D3 received arthritogenic thoracic duct cells and were monitored for severity of arthritis. CD45(+) cells obtained by collagenase digestion of hind-paw synovium-rich tissues (SRTs) were analysed to observe the effects of dietary vit D3 on the inflammatory process. Arthritis was more severe in vitamin D-deficient (vit-D(-)) rats compared with vitamin D-replete (vit-D(+)) rats. Resolution was delayed in vit-D(-) rats compared with vit-D(+) rats, or rats fed standard chow. During the acute phase of ATA, numbers of CD45(+) cells were significantly increased in the SRTs of vit-D(-) rats compared with vit-D(+) rats. This increase involved T-cells, polymorphonuclear leukocytes, macrophages, dendritic cells (DCs) and MHC II(hi) cells that resemble activated monocytes. A major difference between the dietary groups was that most DCs at the peak of inflammation in vit-D(-) rats were CD4(-), whereas in convalescent vit-D(+) rats most expressed CD4. Multiple categories of genes expressed by DCs differed between deficient and replete rats, with deficiency being associated with relative upregulation of certain pro-inflammatory genes and replete status being associated with upregulation of genes associated with resolution of inflammation. The findings indicate that ATA is more severe and prolonged in vit-D deficiency, that vit-D deficiency promotes accumulation of CD4(-) DCs in synovium during ATA and that a gene-expression profile is likely to contribute to the observed increased severity and duration of arthritis.

Adoptive transfer of peripheral immune cells potentiates allodynia in a graded chronic constriction injury model of neuropathic pain.

Recent evidence demonstrates that peripheral immune cells contribute to the nociceptive hypersensitivity associated with neuropathic pain by infiltrating the central nervous system (CNS). We have recently developed a rat model of graded chronic constriction injury (CCI) by varying the exposure of the sciatic nerve and control non-nerve tissue to surgical placement of chromic gut. We demonstrate that splenocytes can contribute significantly to CCI-induced allodynia, as adoptive transfer of these cells from high pain donors to low pain recipients potentiates allodynia (P<0.001). The phenomenon was replicated with peripheral blood mononuclear cells (P<0.001). Adoptive transfer of allodynia was not achieved in sham recipients, indicating that peripheral immune cells are only capable of potentiating existing allodynia, rather than establishing allodynia. As adoptively transferred cells were found by flow cytometry to migrate to the spleen (P<0.05) and potentiation of allodynia was prevented in splenectomised low pain recipients, adoptive transfer of high pain splenocytes may induce the migration of host-derived immune cells from the spleen to the CNS as observed by flow cytometry (P<0.05). Importantly, intrathecal transfer of CD45(+) cells prepared from spinal cords of high pain donors into low pain recipients led to potentiated allodynia (P<0.001), confirming that infiltrating immune cells are not passive bystanders, but actively contribute to nociceptive hypersensitivity in the lumbar spinal cord.

Telomere length in lymphocytes of older South Australian men may be inversely associated with plasma homocysteine.

Deficiencies in folate (FOL) and vitamin B12 (B12) result in increased chromosomal aberrations, a validated biomarker of cancer risk. Telomeres, the regions of DNA that cap the ends of each chromosome, are critical for maintaining chromosomal stability but the impact of micronutrients on telomere structure and function remains unclear. We hypothesized that telomere length maintenance might be compromised if the status of FOL and B12 was inadequate and plasma homocysteine (HCY) was increased. We investigated the relationship between telomere length in peripheral blood lymphocytes and plasma FOL, B12, and HCY status, and tested whether any such relationship was dependent on age, gender, body mass index, and common polymorphisms in folate metabolism genes. A single blood sample was collected from 43 younger (18-32 years) and 47 older (65-83 years) adults in South Australia. The younger cohort consisted of 18 males and 25 females, whereas the older group included 24 males and 23 females. Telomere length was determined in lymphocytes by flow cytometry. Telomere length in the younger cohort was 11.52% greater than in the older cohort (p = 0.015). In the older cohort, telomere length in females was 12.5% greater than in males (p = 0.028). In older males, there was a significant inverse correlation between telomere length and HCY (r = -0.57, p = 0.004), but this effect was not observed in the younger cohort or in the older female group. These results provide evidence that telomere length of lymphocytes in older men may be adversely affected by HCY in vivo.

Recent thymic origin, differentiation, and turnover of regulatory T cells.

Regulatory CD4+ T cells (Treg) are essential to maintain self-tolerance. Release of natural Treg from the thymus is believed to commence soon after birth, but it is unclear how many are produced by "conversion" in the periphery, whether numbers are maintained after puberty by general homeostatic mechanisms that regulate lymphocyte numbers, or whether significant numbers are produced by the involuted thymus. To address the origin of Treg in normal adult rats, we focused on recent thymus emigrants (RTE). Approximately 30% of CD4+CD25+forkhead box p3 (Foxp3)+ Treg expressed markers associated with RTE. Following thymectomy, numbers of cells expressing these markers fell by 80% within 30 days. Furthermore, although only approximately 5% of CD4+ single-positive thymocytes expressed Foxp3 within 24 h after intrathymic injection of FITC, more than 30% of the labeled CD4+ RTE were Foxp3+, suggesting that some RTE may acquire Foxp3 in the periphery. Thus, some RTE may acquire Foxp3 rapidly after emigration from the thymus. Treg are dividing rapidly with apparent half-lives of approximately 18 days and approximately 7 days for the CD4+CD25+Foxp3+ and CD4+CD25-Foxp3+ subsets, respectively. The apparently slower turnover of CD4+CD25+Foxp3+ cells is a result of CD4+CD25+Foxp3+ --> CD4+CD25-Foxp3+ conversion, with no loss of regulatory function. Taken together, the data suggest that Treg in adults are relatively short-lived and that their numbers are maintained by rapid cell division and continuous replenishment from the thymus.

Importance of NKT cells in resistance to herpes simplex virus, fate of virus-infected neurons, and level of latency in mice.

Herpes simplex virus type 1 (HSV-1) produces acute mucocutaneous infections, spread to sensory ganglia, and establishment of latency. In addition, neurovirulent strains have potential to invade the central nervous system (CNS), with potentially a lethal outcome. Early activation of defenses at all stages is essential to limit virus load and reduce the risk of neuronal damage, extensive zosteriform skin lesions, and catastrophic spread to the CNS. NKT cells respond rapidly, and we have shown previously that CD1d-deficient mice are compromised in controlling a neuroinvasive isolate of HSV-1. We now compare infection in Jalpha18 GKO and CD1d GKO mice, allowing direct assessment of the importance of invariant Valpha14(+) NKT cells and deduction of the role of the CD1d-restricted NKT cells with diverse T-cell receptors. The results indicate that both subsets of NKT cells contribute to virus control both in the afferent phase of infection and in determining the mortality, neuroinvasion, loss of sensory neurons, size of zosteriform, lesions and levels of latency. In particular, both are crucial determinants of clinical outcome, providing protection equivalent to a 1-log dose of virus. These NKT cells can be expected to provide protection at doses of virus that might be encountered naturally.

The Src/ABL kinase inhibitor dasatinib (BMS-354825) inhibits function of normal human T-lymphocytes in vitro.

Dasatinib (BMS-354825) is a Src/ABL tyrosine kinase inhibitor currently approved for the treatment of chronic myeloid leukemia. Dasatinib has increased potency against ABL compared to the current therapy imatinib, and is effective in many cases where disease is resistant to imatinib. Dasatinib also inhibits many Src-family tyrosine kinases. We have demonstrated in this study that dasatinib is able to block the function of normal human T-lymphocytes in vitro at clinically relevant concentrations. T-cell functions including proliferation, activation and cytokine production were all uniformly inhibited in the presence of dasatinib. We also demonstrated inhibition of TCR signalling through Src-family kinase LCK, and predicted that inhibition of LCK and other kinases involved in T-cell signalling by dasatinib is responsible for the suppression of T-cell function. These findings raise the concern about potential T-cell inhibition in patients taking dasatinib, and suggest a possible application for the treatment of T-cell mediated immune disorders.

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate.

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. We propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.

Expression of a B-cell-restricted isoform of CD45 is associated with maturity in rat serosal and connective-tissue mast cells.

Fas-associated protein with death domain/mediator of receptor induced toxicity (FADD/MORT1) was first described as a transducer of death receptor signalling but was later recognized also to be important for proliferation of T cells. B-cell lymphoma 3 (Bcl-3) is a relatively little understood member of the nuclear factor (NF)-kappaB family of transcription factors. We recently found that Bcl-3 is up-regulated in T cells from mice where FADD function is blocked by a dominant negative transgene (FADD-DN). To understand the importance of this, we generated FADD-DN/bcl-3(-/-) mice. Here, we report that T cells from these mice show massive cell death and severely reduced proliferation in response to T-cell receptor (TCR) stimulation in vitro. Transgenic coexpression of Bcl-2 (FADD-DN/bcl-3(-/-)/vav-bcl-2 mice) rescued the survival but not the proliferation of T cells. FADD-DN/bcl-3(-/-) mice had normal thymocyte numbers but reduced numbers of peripheral T cells despite an increase in cycling T cells in vivo. However, activation of the classical NF-kappaB and extracellular regulated kinase (ERK) pathways and expression of interleukin (IL)-2 mRNA upon stimulation were normal in T cells from FADD-DN/bcl-3(-/-) mice. These data suggest that FADD and Bcl-3 regulate separate pathways that both contribute to survival and proliferation in mouse T cells.

Recruitment of dendritic cells and macrophages during T cell-mediated synovial inflammation.

Adoptive transfer of adjuvant-induced arthritis was used in this study to examine local macrophages and dendritic cells (DCs) during T cell-mediated synovial inflammation. We studied the influx of CD11b+CD11c+ putative myeloid DCs and other non-lymphoid CD45+ cells into synovium-rich tissues (SRTs) of the affected hind paws in response to a pulse of autoreactive thoracic duct cells. Cells were prepared from the SRTs using a collagenase perfusion-digestion technique, thus allowing enumeration and phenotypic analysis by flow cytometry. Numbers of CD45+ cells increased during the first 6 days, with increases in CD45+MHC (major histocompatibility complex) II+ monocyte-like cells from as early as day 3 after transfer. In contrast, typical MHC II(-) monocytes, mainly of the CD4(-) subset, did not increase until 12 to 14 days after cell transfer, coinciding with the main influx of polymorphonuclear cells. By day 14, CD45+MHC IIhi cells constituted approximately half of all CD45+ cells in SRT. Most of the MHC IIhi cells expressed CD11c and CD11b and represented putative myeloid DCs, whereas only approximately 20% were CD163+ macrophages. Less than 5% of the MHC IIhi cells in inflamed SRT were CD11b(-), setting a maximum for any influx of plasmacytoid DCs. Of the putative myeloid DCs, a third expressed CD4 and both the CD4+ and the CD4(-) subsets expressed the co-stimulatory molecule CD172a. Early accumulation of MHC IIhiCD11c+ monocyte-like cells during the early phase of T cell-mediated inflammation, relative to typical MHC II(-) blood monocytes, suggests that recruited monocytes differentiate rapidly toward the DC lineage at this stage in the disease process. However, it is possible also that the MHC IIhiCD11c+ cells originate from a specific subset of DC-like circulating mononuclear cells.

Importance of the carboxyl terminus of FAT/CD36 for plasma membrane localization and function in long-chain fatty acid uptake.

This study investigates the role of the cytoplasmic C terminus of fatty acid translocase (FAT/CD36) in localization of the molecule to the plasma membrane, its insertion into lipid rafts, and its ability to enhance long-chain fatty acid uptake in transfected H4IIE rat hepatoma cells. In these cells, wild-type FAT/CD36 is localized to both lipid raft and nonraft domains of the plasma membrane. Interestingly, a FAT/CD36 truncation mutant lacking the final 10 amino acids of the cytoplasmic C terminus was retained within the cell in detergent-resistant membranes, and unlike wild-type FAT/CD36, it did not enhance oleate uptake. Furthermore, expression of FAT/CD36 in these cells increased the incorporation of oleate into diacylglycerol, a property that was not shared by truncated FAT/CD36. To examine whether the C terminus itself has an intrinsic ability to dictate the plasma membrane localization of FAT/CD36, this region was fused in-frame to enhanced green fluorescent protein (EGFP). This domain was sufficient to attach EGFP to cellular membranes, suggesting an involvement in the intracellular traffic of the molecule. We conclude that the C terminus of FAT/CD36 is required for localization of the receptor to the cell surface and its ability to enhance cellular oleate uptake.

Recruitment and proliferation of CD4+ T cells in synovium following adoptive transfer of adjuvant-induced arthritis.

Adjuvant-induced arthritis can be transferred to naive Dark Agouti (DA) strain (DA.CD45.1) rats by thoracic duct (TD) lymphocytes. Disease can be re-induced in convalescent rats by further transfer of arthritogenic cells, suggesting that resolution of the adoptive disease is not due to active regulation. To examine whether resolution is due to exhaustion of effector cells, we transferred the disease to DA.CD45.1 recipients, using CD4+ T cells from DA.CD45.2 donors. At the height of the adoptively transferred disease, donor cells comprised only 5-10% of recirculating CD4+ T cells but they accounted for approximately 40% of the CD4+ T cells in synovium-rich tissues of the hind paws. Approximately 65% of the donor cells in the synovium expressed a marker of proliferation (Ki-67 antigen). Division of CD4+ T cells continued in shielded paws after suppression of the recirculating pool of lymphocytes by selective irradiation. Intravenously injected CD4+ TD T lymphoblasts from arthritic donors were recruited to normal paws and, in greater numbers, to paws of animals with existing arthritis. Survival of the [125I]iodo-deoxyuridine-labeled lymphoblasts was greater in animals with existing arthritis. We conclude that effector CD4+ T cells in target tissues can proliferate in response to autoantigens and exhibit enhanced survival. However, without a continuous supply, adoptively transferred effector cells do not produce autonomous local disease, due to limits to their lifespan and ability to replicate indefinitely.

MHC II+ CD45+ cells from synovium-rich tissues of normal rats: phenotype, comparison with macrophage and dendritic cell lineages and differentiation into mature dendritic cells in vitro.

Synovial tissues are frequent sites of inflammatory disorders in which dendritic cells (DCs) may play an important role. This study examines potential antigen-presenting cells obtained from synovium-rich tissues (SRTs) by vascular perfusion of rat hind limbs with collagenase and further enzymatic digestion of the disarticulated hind paws in vitro. The three sub-populations of interest were: CD45+MHC IIhi, mainly CD11c+ and CD163-; CD45+MHC IIlo, mainly CD11c- and CD163+ and CD45+MHC II-, mainly CD11c- and CD163+. Expression of CD11c and CD163 correlated with ruffled cell-surface (CD11c+CD163-) and highly vacuolated cytoplasm (CD11c-CD163+), respectively. Culture of the CD45+CD163- sub-population in granulocyte macrophage colony-stimulating factor (GM-CSF) yielded CD45+MHC IIhi CD11c+CD163- cells with veiled morphology, while the large vacuolated cells that expressed CD163 resembled type A synoviocytes in both surface antigen phenotype and morphology. These results demonstrate that SRTs contain indeterminate cells that can differentiate into mature DCs in vitro in response to GM-CSF, plus mature synovial lining macrophages.

MHC class II compartment, endocytosis and phagocytic activity of macrophages and putative dendritic cells isolated from normal tissues rich in synovium.

The endocytic and phagocytic activities of a population of MHC IIhi CD11c+ dendritic cell (DC)-like cells in synovium-rich tissues (SRTs) of normal rat paws were compared with CD163+ cells (putative macrophages) from the same tissues and pseudo-afferent lymph DCs, peritoneal macrophages and blood monocytes. Fifty percent of CD11c+ cells and 75% of CD163+ cells isolated from SRT internalized fluorescein-conjugated dextran (FITC-DX). Of these endocytic cells, half of those expressing CD11c, but only 30% of those expressing CD163, were surface MHC class II+ (sMHC II+). CD11c+ cells were more endocytic than monocytes or pseudo-afferent lymph DC, but some CD163+ cells (type A synoviocytes) were found to be highly endocytic. CD163+ cells from SRT were more phagocytic (25%) than the general MHC class II+ population (16%). Of phagocytic cells, 40% of CD163+ cells were sMHC II(variable) and they constituted 60% of all MHC class II+ phagocytic cells. Only 18% of phagocytic MHC II+ cells expressed CD11c and the most of these were MHC IIhi. In comparison, 60% of CD163+ peritoneal macrophages were phagocytic, while blood monocytes were poorly phagocytic. Intracellular MHC class II-rich compartments (MIIC) were prominent in sMHC IIhi cells in SRT but rare in CD163+ cells. Most MHC IIhi CD11c+ cells did not have a detectable MIIC.

Blockade of IFN-gamma does not affect the arthritogenicity of T cells generated during the induction of adjuvant arthritis but exacerbates the polyarthritis produced by adoptive transfer of arthritogenic effector cells.

IFN-gamma production is prominent in some models of autoimmune disease, including adjuvant arthritis (AA), but the role of IFN-gamma in the pathogenesis of these diseases is uncertain. Experimental manipulation (administration of cytokine, blocking cytokine action with specific antibody, disruption of genes encoding the cytokine or its receptor) has revealed both pro- and anti-inflammatory effects, depending on the nature of the manipulation and the timing of the treatment. We examined separately the effects of cytokine blockade during the afferent and efferent phases of AA in Dark Agouti rats, using an adoptive transfer system. Effects of IFN-gamma on the efferent phase were investigated by treating recipients with mAb DB-1, which blocks the activity of rat IFN-gamma. When treatment was commenced before cell transfer, the resulting polyarthritis was more severe than in controls treated with normal IgG. Commencing treatment after the adoptively transferred disease had become established caused neither amelioration nor exacerbation, but did cause some delay in resolution. In contrast, treatment of donors did not appear to affect the generation of arthritogenic cells. The main effect of IFN-gamma appears to be modulation of the arthritogenicity of the migratory effector T cells that can transfer AA.

Origin of macrophages in a kaolin-induced model of rat syringomyelia: a study using radiation bone marrow chimeras.

STUDY DESIGN: Animal experimental study. OBJECTIVE: To study the origin of macrophages in a rat model of syringomyelia. SUMMARY OF BACKGROUND DATA: Syringomyelia is a clinically important condition in which a cystic cavity forms within the spinal cord. This leads to significant delayed neurologic deterioration, which may be manifested as weakness, numbness, or pain. The pathophysiology and mechanism of syrinx formation remain unclear. Human autopsy findings have demonstrated a prominent accumulation of macrophages in relation to the syrinx. Similar observations have also been made in a previously established rat model of syringomyelia. Little is known about the origin and precise functions of these cells. METHODS: Syrinx formation was induced by intraparenchymal injections of kaolin within the cervical spinal cords of 30 DA rat (RT7.1) radiation bone marrow chimeras reconstituted with bone marrow from RT7.2 congeneic donors. The distribution of macrophages was evaluated at survival times of 3 days, 1 week, and 4 weeks. Immunostaining of fresh-frozen spinal cord tissue was performed using specific antibodies against rat macrophage ED1 antigen and RT7.2 allele of CD45. This allowed donor-derived hematogenous (ED1+, RT7.2+) macrophages to be distinguished from native cells (ED1+, RT7.2-). RESULTS: Central canal dilatation was seen from 1 week. This was associated with extensive accumulation of ED1+ macrophages within the spinal cord parenchyma. A large influx of bone marrow-derived (ED1+, RT7.2+) macrophages was observed. However, a considerable proportion of resident microglia (RT7.2-) also upregulated ED1. These activated microglia demonstrated distinct morphologic features. CONCLUSIONS: Large numbers of macrophages were recruited from the bone marrow in kaolin-induced rat syringomyelia. However, a significant number of resident microglia upregulated their ED1 activity and appear to provide a substantial source of macrophages.

Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin.

A genetic analysis of Giardia intestinalis, a parasitic protozoan species that is ubiquitous in mammals worldwide, was undertaken using organisms derived from a variety of mammalian hosts in different geographical locations. The test panel of 53 Giardia isolates comprised 48 samples of G. intestinalis, including representatives of all known genetic subgroups, plus an isolate of G. ardeae and four isolates of G. muris. The isolates were compared by allozymic analysis of electrophoretic data obtained for 21 cytosolic enzymes, representing 23 gene loci. Neighbour Joining analysis of the allelic profiles supported the monophyly of G. intestinalis but showed that the species encompasses a rich population substructure. Seven major clusters were evident within G. intestinalis, corresponding to lineages designated previously as genetic assemblages A-G. Some genotypes, e.g. those defining assemblage A, are found in divergent host species and may be zoonotic. However other genotypes, e.g. those defining assemblages C-G, appear to be confined to particular hosts or host groups. The findings reinforce other evidence that G. intestinalis, which was defined on the basis of morphological criteria only, is a species complex.