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Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule–driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated. Key Points: • Aged erythrocytes interact with the extracellular matrix of the spleen, resulting in hemolysis and ghost formation. • Ghost formation enables recognition and phagocytosis of senescent erythrocytes by RPMs.

Background: The need for rapid and accurate comparison of panels of adjuvanted vaccine formulations and subsequent rational down selection, presents several challenges for modern vaccine development. Here we describe a method which may enable vaccine and adjuvant developers to compare antigen/adjuvant combinations in a harmonized fashion. Three reference antigens: Plasmodium falciparum apical membrane antigen 1 (AMA1), hepatitis B virus surface antigen (HBsAg), and Mycobacterium tuberculosis antigen 85A (Ag85A), were selected as model antigens and were each formulated with three adjuvants: aluminium oxyhydroxide, squalene-in-water emulsion, and a liposome formulation mixed with the purified saponin fraction QS21.Results: The nine antigen/adjuvant formulations were assessed for stability and immunogenicity in mice in order to provide benchmarks against which other formulations could be compared, in order to assist subsequent down selection of adjuvanted vaccines. Furthermore, mouse cellular immune responses were analyzed by measuring IFN-γ and IL-5 production in splenocytes by ELISPOT, and humoral responses were determined by antigen-specific ELISA, where levels of total IgG, IgG1, IgG2b and IgG2c in serum samples were determined.Conclusions: The reference antigens and adjuvants described in this study, which span a spectrum of immune responses, are of potential use as tools to act as points of reference in vaccine development studies. The harmonized methodology described herein may be used as a tool for adjuvant/antigen comparison studies.

Phenotypic variation of quantitative traits is orchestrated by a complex interplay between the environment (e.g. diet) and genetics. However, the impact of gene-environment interactions on phenotypic traits mostly remains elusive. To address this, we feed 1154 mice of an autoimmunity-prone intercross line (AIL) three different diets. We find that diet substantially contributes to the variability of complex traits and unmasks additional genetic susceptibility quantitative trait loci (QTL). By performing whole-genome sequencing of the AIL founder strains, we resolve these QTLs to few or single candidate genes. To address whether diet can also modulate genetic predisposition towards a given trait, we set NZM2410/J mice on similar dietary regimens as AIL mice. Our data suggest that diet modifies genetic susceptibility to lupus and shifts intestinal bacterial and fungal community composition, which precedes clinical disease manifestation. Collectively, our study underlines the importance of including environmental factors in genetic association studies., Complex traits associate with genetic variation and environment and their interaction. Here, the authors study the influence of different diets on trait variability in 1154 outbred mice from an advanced intercross line and find gene-diet interactions associated with spontaneous autoimmunity development in these animals.

T and B cell compartmentalization is a hallmark of secondary lymphoid organs and is maintained by chemokine-expressing stromal cells. How this stromal cell network initially develops and differentiates into two distinct subsets is poorly known, especially for the splenic white pulp (WP). Here, we show that perivascular fibroblast precursors are triggered by LTα1β2 signals to expand, express CCL19/21, and then differentiate into two functionally distinct fibroblast subsets responsible for B and T cell clustering and WP compartmentalization. Failure to express or sense CCL19 leads to impaired T zone development, while lack of B cells or LTα1β2 leads to an earlier and stronger impairment in WP development. We therefore propose that WP development proceeds in multiple steps, with LTα1β2 + B cells acting as major inducer cells driving the expansion and gradual differentiation of perivascular fibroblasts into T and B zone organizer cells.

Objective— Coronary heart disease is associated with monocytosis. Studies using animal models of monocytosis and atherosclerosis such as ApoE −/− mice have shown bone marrow (BM) hematopoietic stem and multipotential progenitor cell (HSPC) expansion, associated with increased cell surface expression of the common β subunit of the granulocyte macrophage colony–stimulating factor/interleukin-3 receptor (CBS) on HSPCs. ApoE −/− mice also display increased granulocyte macrophage colony–stimulating factor–dependent monocyte production in the spleen. We investigated the role of the CBS in cholesterol-driven HSPC expansion, monocytosis, and atherosclerosis. Approach and Results— Ldlr −/− mice were transplanted with ApoE −/− Cbs −/− or ApoE −/− BM followed by Western-type diet feeding. Compared with ApoE −/− BM–transplanted controls, ApoE −/− Cbs −/− BM–transplanted mice had reduced BM and splenic HSPC proliferation, fewer blood monocytes and neutrophils, and reduced macrophage content and area of early atherosclerotic lesions. More advanced lesions showed diminished macrophage and collagen content; however, lesion size was unchanged, reflecting an increase in necrotic core area, associated with a marked decrease in Abcg1 expression and increased macrophage apoptosis. Compared with wild-type mice, Western-type diet–fed ApoE −/− mice showed increased CBS expression on granulocyte macrophage colony–stimulating factor–producing innate response activator B cells and expansion of this population. ApoE −/− Cbs −/− BM–transplanted Ldlr −/− mice showed a marked decrease in innate response activator B cells compared with ApoE −/− BM–transplanted Ldlr −/− controls. Conclusions— Increased levels of CBS on HSPCs and splenic innate response activator B cells lead to expansion of these populations in ApoE −/− BM–transplanted Ldlr −/− mice, contributing to monocytosis and increased lesional macrophage content. However, in more advanced lesions, the CBS also has a role in atherosclerotic plaque stabilization.

Rationale: Inflamed atherosclerotic plaques can be visualized by noninvasive positron emission and computed tomographic imaging with 18 F-fluorodeoxyglucose, a glucose analog, but the underlying mechanisms are poorly understood. Objective: Here, we directly investigated the role of Glut1-mediated glucose uptake in apolipoprotein E–deficient ( ApoE −/− ) mouse model of atherosclerosis. Methods and Results: We first showed that the enhanced glycolytic flux in atheromatous plaques of ApoE −/− mice was associated with the enhanced metabolic activity of hematopoietic stem and multipotential progenitor cells and higher Glut1 expression in these cells. Mechanistically, the regulation of Glut1 in ApoE −/− hematopoietic stem and multipotential progenitor cells was not because of alterations in hypoxia-inducible factor 1α signaling or the oxygenation status of the bone marrow but was the consequence of the activation of the common β subunit of the granulocyte-macrophage colony-stimulating factor/interleukin-3 receptor driving glycolytic substrate utilization by mitochondria. By transplanting bone marrow from WT, Glut1 +/− , ApoE −/− , and ApoE −/− Glut1 +/− mice into hypercholesterolemic ApoE-deficient mice, we found that Glut1 deficiency reversed ApoE −/− hematopoietic stem and multipotential progenitor cell proliferation and expansion, which prevented the myelopoiesis and accelerated atherosclerosis of ApoE −/− mice transplanted with ApoE −/− bone marrow and resulted in reduced glucose uptake in the spleen and aortic arch of these mice. Conclusions: We identified that Glut1 connects the enhanced glucose uptake in atheromatous plaques of ApoE −/− mice with their myelopoiesis through regulation of hematopoietic stem and multipotential progenitor cell maintenance and myelomonocytic fate and suggests Glut1 as potential drug target for atherosclerosis.

A cDNA encoding the chicken homologue of the human myelomonocytic differentiation antigen, CD14, was cloned by RT-PCR from chicken bone marrow cell RNA, using oligonucleotide primers based on the predicted cDNA sequence. The cloned chicken CD14 (chCD14) cDNA encodes an open reading frame of 465 amino acids (aa), with 31-34% aa identity to mouse, bovine and human (hu) CD14. As in mouse and man, chCD14 is a leucine-rich protein. In mammals, CD14 is a GPI-anchored protein. Protein structure analysis suggested that chCD14, by contrast, was potentially a trans-membrane protein. The predicted aa sequence comprises an extracellular domain of 417 aa, followed by a 23-aa trans-membrane segment, and a 25-aa intracytoplasmic region, the latter containing no obvious signalling motifs. COS-7 cells were transfected with p3XFLAG-CMV-8::chCD14 or pCDM8::huCD14, incubated with or without PI-PLC and stained with anti-FLAG or anti-huCD14 antibody respectively. PI-PLC cleaved huCD14 but not chCD14, suggesting that chCD14 is not GPI-anchored. Real-time quantitative RT-PCR analysis revealed that chCD14 mRNA was expressed in most lymphoid and non-lymphoid tissues, except muscle. ChCD14 mRNA was also expressed in most cells examined but strongly expressed in chicken peripheral blood monocyte/macrophages and KUL01+ splenocytes.

We developed an easy and fast method to isolate extracellular matrix tenascin-X (TNX) from various tissues in mice based on TNX antibody affinity purification. We purified approximately 350-kDa cellular interstitial TNX (iTNX) from the spleen, liver and kidney as well as 200-kDa serum TNX (sTNX). Since the nature and significance of glycosylation in TNX remains to be elucidated, glycobiochemical properties of purified TNX were characterized by lectin blot analysis. Lectin blots by Con A, LCA, PHA-E4, RCA120 or WGA revealed the presence of N-glycan in the cellular TNX and especially complex-type N-glycan in the serum TNX. In addition, the iTNX from liver and kidney also possessed O-glycan based on the reaction to PNA. The binding to AAL indicated that iTNX from the three tissues possesses fucose linked alpha1,6 to a pentasaccharide core, whereas sTNX does not. The reaction to SSA but not to MAM suggested the presence of sialic acid linked alpha2,6 to galactose in both cellular and serum TNX. Lectin blots of trypsin-treated iTNX from the spleen also demonstrated that WGA alone reacts to the t300 product derived from the amino-terminal 300-kDa portion.

C1q deficiency in both humans and mice is strongly associated with autoimmunity. We have previously shown that bone marrow transplantation (BMT) restored C1q levels in C1q-deficient (C1qa(-/-)) mice. Here, we studied the effect of BMT on autoimmunity in C1qa(-/-) mice. Following irradiation, young C1qa(-/-) or wild-type MRL/Mp mice received bone marrow cells (BMC) from strain-matched wild-type or C1qa(-/-) animals. C1q levels increased rapidly when C1qa(-/-) mice received BMC from wild-type mice. Conversely, they decreased slowly in wild-type mice transplanted with C1qa(-/-) BMC. C1qa(-/-) animals transplanted with C1qa(-/-) BMC demonstrated accelerated disease when compared with wild-type mice given wild-type BMC. In contrast, a significant delay in the development of autoantibodies and glomerulonephritis was observed in C1qa(-/-) mice reconstituted with wild-type BMC, and the impaired clearance of apoptotic cells, previously described in C1qa(-/-) mice, was rectified. Moreover, the autoimmune disease was accelerated in wild-type mice given C1qa(-/-) BMC compared to animals transplanted with wild-type cells. These results provide supporting evidence that BMT may be a therapeutic option in the treatment of autoimmunity associated with human C1q deficiency.

The obese strain (OS) of chickens, which suffers from spontaneous autoimmune thyroiditis, is an excellent animal model for Hashimoto’s thyroiditis and provides a unique opportunity to investigate the mechanisms underlying and driving the onset of the disease. Following recent advances in cloning chicken cytokines, we can now begin to investigate the role of cytokines in driving the lymphoid infiltration of the thyroid seen in these birds from day 7 posthatch. Using real-time quantitative RT-PCR, we characterized the expression of IFN-γ, IL-1β, IL-2, IL-6, IL-8, IL-15, and IL-18 in thyroids from OS birds and control CB line birds, both in the embryo just before hatch (embryonic day 20) and at 3 and 5 days posthatch. All of these cytokines were up-regulated compared with levels in thyroids from CB birds, at least at some time points, with some evidence for coordination of regulation, e.g., for the proinflammatory cytokines IL-1β and IL-8. Only IL-15 was up-regulated at all time points. IL-15 was also shown to be up-regulated in spleens of OS birds at embryonic day 20 and 5 days posthatch, suggesting that IL-15 is constitutively up-regulated in this line of birds. This could explain the general immune system hyperreactivity exhibited by OS chickens and may be a factor driving the lymphoid infiltration of the thyroid.

Injection of 10 cysts of Toxoplasma gondii (Me49 strain) into Swiss Webster mice results in 1) an acute phase of infection lasting for 2–3 wk, characterized by weight loss, and 2) a chronic phase in which surviving mice show either partial weight recovery (Gainers) or persistent, although stable, cachexia (Nongainers). In response to a second immunological stimulation with lipopolysaccharide (LPS) in the chronic phase of the infection, it is shown that 1) the increase in energy expenditure was more prolonged in both groups of infected mice than in controls, 2) the intensity and duration of hypophagia were also differently affected with Nongainers > Gainers > controls, and 3) the infected mice had higher serum levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-10 and a lower ratio of IL-10 to TNF-α than controls. In contrast, serum IL-4 increased to the same level in all three groups. Evaluation of the permeability of the blood-brain barrier by intravenous injection of Evans blue revealed a marked staining in the brain of only the infected Nongainers. Taken together, these results indicate that, in mice with chronic toxoplasmosis, a second nonspecific challenge (with LPS) exacerbates the hypophagic and hypermetabolic states, the latter being associated with hyperresponsiveness in TNF-α and IL-10 production. Furthermore, the greater exacerbation of the hypophagic state in mice showing persistent cachexia may be due to a preexisting higher permeability of the blood-brain barrier, which would allow a greater access of plasma-borne cytokines and/or other neuroimmunologically active substances to the central nervous system.

Homogenates of chick dorsal root ganglia (DRG) and in vitro cultures of DRG neurons are known to synthesize prostaglandin (PG) D2. To specify the PGD synthase isozymes controlling PGD2 synthesis in DRG and to identify the DRG cells responsible for this synthesis, we applied polyclonal antibodies raised against rat brain or rat spleen PGD synthase isozymes to vibratome or cryostat slices of DRG previously fixed with a formaldehyde-lysine-periodate mixture and permeabilized with Triton X-100. The immunoreactivity indicating rat spleen PGD synthase, a glutathione (GSH)-requiring enzyme, was located in satellite cells encompassing particular large neurons of class A and in Schwann cells myelinating and enwrapping their initial axonal segments. In contrast, the immunoreactivity of rat brain PGD synthase, a GSH- independent enzyme, was restricted to particular ganglion cell perikarya: 33% of the DRG neurons were immunostained for rat brain PGD synthase, including 2% of large class A neurons and 40% of small class B neurons. Only 3.3% of rat brain PGD synthase-immunoreactive small B neurons coexpressed substance P, indicating that the immunoreactive neurons belong to the B1 subclass. By electron microscopy, 71 of 72 immunoreactive DRG cells were identified as small B neurons of the B1 subclass, and 71 of 77 B1 neurons were immunoreactive for rat brain PGD synthase. These results demonstrate that PGD2 formation in DRG is regulated by two isozymes: the GSH-requiring isozyme located in satellite and Schwann cells and the GSH-independent isozyme-confined to small B1 neurons.

Background Natural killer T cells represent a linkage between innate and adaptive immunity. They are a heterogeneous population of specialized T lymphocytes composed of different subsets. DX5+NKT cells are characterized by expression of the NK cell marker DX5 in the context of CD3. However, little is known about the phenotype and functional capacity of this unique cell population. Therefore, we investigated the expression of several T cell and NK cell markers, as well as functional parameters in spleen and liver subsets of DX5+NKT cells in NK1.1- Balb/c mice and compared our findings to NK1.1+ C57Bl/6 mice. Results In the spleen 34% of DX5+NKT cells expressed CD62L and they up-regulated the functional receptors CD154 as well as CD178 upon activation. In contrast, only a few liver DX5+NKT cells expressed CD62L, and they did not up-regulate CD154 upon activation. A further difference between spleen and liver subsets was observed in cytokine production. Spleen DX5+NKT cells produced more Th1 cytokines including IL-2, IFN-γ and TNF-α, while liver DX5+NKT cells secreted more Th2 cytokines (e.g. IL-4) and even the Th17 cytokine, IL-17a. Furthermore, we found inter-strain differences. In NK1.1+ C57Bl/6 mice DX5+NKT cells represented a distinct T cell population expressing less CD4 and more CD8. Accordingly, these cells showed a CD178 and Th2-type functional capacity upon activation. Conclusion These results show that DX5+NKT cells are a heterogeneous population, depending on the dedicated organ and mouse strain, that has diverse functional capacity.

IL-1R antagonist (IL-1Ra) is a natural inhibitor of the pleiotropic proinflammatory activities of IL-1. Although several reports described the effects of complete IL-1Ra deficiency, no study has examined the consequences of cell type-specific IL-1Ra inactivation during systemic inflammation. Previous in vitro data demonstrated high IL-1Ra production by hepatocytes and myeloid cells after endotoxin stimulation. In addition, hepatocyte IL-1Ra production is regulated as an acute-phase protein in vitro. In this study, we analyzed the production and functional role of hepatocyte- and myeloid cell-derived IL-1Ra during endotoxin-induced septic shock and acute IL-1β–induced sterile inflammation. Using conditional IL-1Ra knockout mice, we showed that hepatocytes and myeloid cells are the two major cellular sources of circulating IL-1Ra in response to LPS. Interestingly, IL-1Ra production by myeloid cells, but not hepatocytes, is critical for survival during endotoxemia. Furthermore, we provide the first in vivo evidence demonstrating that IL-1Ra is produced as an acute-phase protein by hepatocytes during IL-1β–induced inflammation and that hepatocyte-derived IL-1Ra functions as an endogenous negative feedback downregulating the proinflammatory effects of IL-1. Taken together, our observations define distinct roles for two major cellular sources of IL-1Ra in response to different types of systemic inflammatory stimuli in vivo.

OBJECTIVE: T cell production of RANKL, interferon-gamma (IFNgamma), and other cytokines in inflammatory processes such as rheumatoid arthritis or secondary to conditions such as estrogen deficiency stimulates osteoclast activity, which leads to bone resorption and bone loss. The purpose of this study was to characterize the effects of interleukin-15 (IL-15), a master T cell growth factor whose role in bone remodeling remains unknown. METHODS: We used mice lacking the IL-15 receptor (IL-15Ralpha(-/-) ) to investigate the effects of IL-15 on osteoclast development, T cell and dendritic cell activation in vitro and in vivo, bone mass, and microarchitecture in intact and ovariectomized (OVX) mice. RESULTS: In wild-type (WT) animals, IL-15 and RANKL provided a costimulatory signal for osteoclast development. Spleens from IL-15Ralpha(-/-) mice contained few c-Kit+ osteoclast precursors, and the expression of NF-ATc1 and the osteoclastogenic response to RANKL were impaired. In addition, dendritic cell-dependent and T cell-dependent mechanisms of osteoclast activation, including RANKL and IFNgamma production, were impaired in IL-15Ralpha(-/-) mice. In turn, IL-15Ralpha(-/-) T cells failed to stimulate WT osteoclasts, whereas WT T cells failed to stimulate IL-15Ralpha(-/-) osteoclasts. Compared with WT mice, both intact and OVX IL-15Ralpha(-/-) mice had significantly greater bone mineral density and microarchitecture, including a higher trabecular bone volume fraction and cortical thickness. The numbers of osteoclasts on the bone surface as well as markers of bone turnover were significantly decreased in IL-15Ralpha(-/-) mice. CONCLUSION: In the absence of IL-15 signaling, several converging mechanisms of osteoclastogenesis are inhibited, both directly and indirectly, through T cells, which leads to a high bone mass phenotype. Targeting the IL-15 pathway may represent a novel therapeutic approach to treating primary and secondary osteoporosis.

A sensitive and specific microELISA assay is described for the immunoactive polypeptide parathymosin. Antibodies against a synthetic peptide corresponding to the rat parathymosin sequence 5-30 were raised in rabbits immunised with this peptide conjugated to keyhole limpet hemocyanin (KLH). The useful range of the assay was 0.25-30 pmol (3-330 ng) of parathymosin and the assay was specific. The related immunoactive polypeptides prothymosin alpha or thymosin alpha 1 showed no cross-reactivity. In spiking experiments the recovery of the assay was found to be greater than 92% at all concentrations tested. The intra-assay variation was 17%, whereas the inter-assay variation was 26%. Using this assay the highest concentration of parathymosin was found in porcine liver, followed by kidney, lung, thymus and spleen. This assay compares favorably with one microELISA and two RIA methods already published, in that it is more sensitive by at least an order of magnitude, and it is simpler and quicker. J Immunol Methods

PURPOSE: To assess functional and morphological aspects of spleen auto-implants and of the splenic inferior pole of rats, post-operatively treated or not with hyperbaric oxygen, as well as the survival of these animals, were studied. METHODS: Seventy-eight male Wistar rats, weighing between 192 and 283 g ( 238,3 ± 9,6g), were randomly distributed into three groups: Group1-(n=20), spleen manipulation; group 2-(n=36), spleen auto-implantation; group3-(n= 22), subtotal splenectomy preserving the inferior pole. Each group was subdivided as follows: subgroup a, not submitted to hyperbaric oxygen therapy: 1a(n=10), 2a(n=21), 3a(n= 13); subgroup b, submitted to the therapy: 1b(n=10), 2b(n=15), 3b(n=9). Blood was collected pre-operatively and 11 days after surgery, for the estimation of lipids and immunoglobulins and the counting of platelets and Howell-Jolly corpuscles. The spleen and remains were taken for histological study. RESULTS: The number of surviving animals was significantly higher in groups 1(p 2. The macro and microscopic appearance in subgroup 2b were more viable than in subgroup 2a, and that of group 3 more viable than in group 2. The survival of the animals carrying their whole spleen or its inferior pole was more frequent than that of the auto-implanted animals. CONCLUSION: Functionality and viability of the whole spleen or of its inferior pole, were better than in the auto-implanted animals. Hyperbaric oxygentherapy contributed to increased survival frequency of auto -implanted animals, and to improve the functionality and viability of the auto-implants and the function of the inferior splenic pole, and did not interfere in animals carrying their whole spleen. OBJETIVO: Estudar aspectos funcionais e morfológicos dos auto-implantes esplênicos e do pólo inferior do baço de ratos, tratados ou não com oxigênio hiperbárico no pós-operatório, e a sobrevida desses animais. MÉTODOS: Foram operados 78 ratos, machos, Wistar, pesando entre 192 g e 283 g ( M.A 238,3 ± 9,6) , distribuídos aleatoriamente em três grupos : 1-(n=20) , manipulação do baço; 2-(n=36), auto-implante esplênico; 3(n= 22), esplenectomia subtotal com preservação do pólo inferior. Cada grupo foi dividido em dois subgrupos: a- não submetido à oxigenoterapia hiperbárica: 1a(n=10), 2a(n=21), 3a(n= 13); b- submetido: 1b(n=10), 2b(n=15), 3b(n=9). O sangue foi colhido para dosagem dos lípides e imunoglobulinas e contagem das plaquetas e dos corpúsculos de Howell-Jolly no pré-operatório e 11 dias após a cirurgia. O baço e os remanescentes foram retirados para estudo histológico. RESULTADOS: O número de animais sobreviventes foi significantemente maior nos grupos 1(p

Transmissible spongiform encephalopathies (TSEs) may be acquired peripherally, in which case infectivity usually accumulates in lymphoid tissues before dissemination to the nervous system. Studies of mouse scrapie models have shown that mature follicular dendritic cells (FDCs), expressing the host prion protein (PrP(c)), are critical for replication of infection in lymphoid tissues and subsequent neuroinvasion. Since FDCs require lymphotoxin signals from B lymphocytes to maintain their differentiated state, blockade of this stimulation with a lymphotoxin beta receptor-immunoglobulin fusion protein (LT beta R-Ig) leads to their temporary dedifferentiation. Here, a single treatment with LT beta R-Ig before intraperitoneal scrapie inoculation blocked the early accumulation of infectivity and disease-specific PrP (PrP(Sc)) within the spleen and substantially reduced disease susceptibility. These effects coincided with an absence of FDCs in the spleen for ca. 28 days after treatment. Although the period of FDC dedifferentiation was extended to at least 49 days by consecutive LT beta R-Ig treatments, this had little added protective benefit after injection with a moderate dose of scrapie. We also demonstrate that mature FDCs are critical for the transmission of scrapie from the gastrointestinal tract. Treatment with LT beta R-Ig before oral scrapie inoculation blocked PrP(Sc) accumulation in Peyer's patches and mesenteric lymph nodes and prevented neuroinvasion. However, treatment 14 days after oral inoculation did not affect survival time or susceptibility, suggesting that infectivity may have already spread to the peripheral nervous system. Although manipulation of FDCs may offer a potential approach for early intervention in peripherally acquired TSEs, these data suggest that the duration of the treatment window may vary widely depending on the route of exposure.

Although the transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases, their agents usually replicate and accumulate in lymphoid tissues long before infection spreads to the central nervous system (CNS). Studies of a mouse scrapie model have shown that mature follicular dendritic cells (FDCs), which express the host prion protein (PrP(c)), are critical for replication of infection in lymphoid tissues. In the absence of mature FDCs, the spread of infection to the CNS is significantly impaired. Tumor necrosis factor alpha (TNF-alpha) secretion by lymphocytes is important for maintaining FDC networks, and signaling is mediated through TNF receptor 1 (TNFR-1) expressed on FDCs and/or their precursors. A treatment that blocks TNFR signaling leads to the temporary dedifferentiation of mature FDCs, raising the hypothesis that a similar treatment would significantly delay the peripheral pathogenesis of scrapie. Here, specific neutralization of the TNFR signaling pathway was achieved through treatment with a fusion protein consisting of two soluble human TNFR (huTNFR) (p80) domains linked to the Fc portion of human immunoglobulin G1 (huTNFR:Fc). A single treatment of mice with huTNFR:Fc before or shortly after intraperitoneal injection with the ME7 scrapie strain significantly delayed the onset of disease in the CNS and reduced the early accumulation of disease-specific PrP in the spleen. These effects coincided with a temporary dedifferentiation of mature FDCs within 5 days of huTNFR:Fc treatment. We conclude that treatments that specifically inhibit the TNFR signaling pathway may present an opportunity for early intervention in peripherally transmitted TSEs.

Hepatic leukemia factor (HLF) is a member of the PAR family of transcription regulatory proteins. We have characterized the rat HLF gene and studied its expression and activity. The rat HLF gene is transcribed from two alternative promoters, alpha and beta, with different circadian amplitudes and tissue specificities. The alpha RNA isoforms produce a 43 kDa protein, HLF43, abundant in brain, liver and kidney, like the previously described human HLF RNA. The beta RNA HLF isoforms use a CUG codon to initiate translation of a novel 36 kDa protein, HLF36, which is shorter at its N-terminus relative to the 43 kDa form. HLF36 is expressed uniquely in the liver, where it is the most abundant HLF protein. Surprisingly, the two proteins accumulate in the liver with different circadian amplitudes and have distinct liver-specific promoter preferences in transfection experiments. Thus, HLF43 stimulates transcription from the cholesterol 7 alpha-hydroxylase promoter much more efficiently than from the albumin promoter, while the converse is true for HLF36.

The liver-enriched transcriptional activator protein DBP accumulates in hepatocytes of adult rats according to a strictly controlled circadian rhythm. DBP is not detectable in liver nuclei during the morning hours. Its level raises sharply during the afternoon and reaches a maximum at about 8 p.m. During the night the cellular DBP concentration decreases below detectability. This oscillation is "free running," transcriptionally regulated, and may be under the negative control of glucocorticoid hormones. In keeping with the rhythmicity of DBP accumulation, the albumin gene, a putative target of DBP, is transcribed more efficiently in the evening than in the morning.

When used in an in vitro transcription assay, the promoter of a cloned alpha 2u globulin gene is much more active in liver than in spleen nuclear extracts. Promoter deletion experiments suggest that both positive and negative regulatory mechanisms may be involved in the differential in vitro transcription from the alpha 2u globulin promoter in these two nuclear extracts. Interestingly, removal of promoter elements upstream from position -74 results in a significant increase of in vitro transcription in spleen but not in liver nuclear extracts, and thus reduces the difference in transcription observed with longer alpha 2u promoters in these two extracts. Deletion of additional nucleotides to position -43 strongly reduces the in vitro transcription efficiency of the promoter in extracts from both tissues. None of the examined promoters containing between 3000 and 22 nucleotides of 5' flanking regions are differentially transcribed in liver nuclear extracts from either male or female rats. Thus, in contrast to cell-type specificity, sex-specificity could not be observed in our in vitro transcription experiments. DNase I protection experiments with crude nuclear extracts and partially or highly purified nuclear proteins suggests the presence of six recognition sites for DNA-binding factors between the TATA element and position -210. Some of these factors could be identified as proteins that also bind to elements within the albumin gene promoter.