Development of CD8alpha-positive dendritic cells from a common myeloid progenitor.

Dendritic cells (DCs) are critical in both initiating adaptive immune responses and maintaining tolerance to self antigens. These apparently contradictory roles have been suggested to depend on different subsets of DCs that arise from either myeloid or lymphoid hematopoietic origins, respectively. Although DC expression of CD8alpha is attributed to a lymphoid origin, here we show that both CD8alpha+ and CD8alpha- DCs can arise from clonogenic common myeloid progenitors in both thymus and spleen. Thus, expression of CD8alpha is not indicative of a lymphoid origin, and phenotypic and functional differences among DC subsets are likely to reflect maturation status rather than ontogeny.

Mecp2 regulates tnfa during zebrafish embryonic development and acute inflammation.

Mutations in MECP2 cause Rett syndrome, a severe neurological disorder with autism-like features. Duplication of MECP2 also causes severe neuropathology. Both diseases display immunological abnormalities that suggest a role for MECP2 in controlling immune and inflammatory responses. Here, we used mecp2-null zebrafish to study the potential function of Mecp2 as an immunological regulator. Mecp2 deficiency resulted in an increase in neutrophil infiltration and upregulated expression of the pro- and anti-inflammatory cytokines Il1b and Il10 as a secondary response to disturbances in tissue homeostasis. By contrast, expression of the proinflammatory cytokine tumor necrosis factor alpha (Tnfa) was consistently downregulated in mecp2-null animals during development, representing the earliest developmental phenotype described for MECP2 deficiency to date. Expression of tnfa was unresponsive to inflammatory stimulation, and was partially restored by re-expression of functional mecp2 Thus, Mecp2 is required for tnfa expression during zebrafish development and inflammation. Finally, RNA sequencing of mecp2-null embryos revealed dysregulated processes predictive for Rett syndrome phenotypes.

Cellular dissection of zebrafish hematopoiesis.

Zebrafish as a model system have been instrumental in understanding early vertebrate development, especially of the hematopoietic system. The external development of zebrafish and their genetic amenability have allowed in-depth studies of multiple blood cell types and their respective genetic regulation. This chapter highlights some new data in zebrafish hematopoiesis regarding primitive and definitive hematopoiesis in the embryonic and adult fish, allowing the isolation of prospective progenitor subsets. It also highlights assays developed to examine the function of these progenitors in vivo and in vitro, allowing an evolutionary understanding of the hematopoietic system and how zebrafish can be better utilized as a model system for a multitude of hematopoietic disorders.

Lack of m2 muscarinic acetylcholine receptor mRNA in rat lymphocytes.

The presence of muscarinic acetylcholine receptors on lymphocytes has been demonstrated by radioligand binding experiments. Although the specific subtype(s) of muscarinic acetylcholine receptors expressed in lymphocytes is still unknown, some reports suggest the presence of the m2 subtype. In this study we analyzed the expression of m2 subtype mRNA in rat mononuclear cells, B lymphocytes and T lymphocytes by Northern blot hybridization and reverse transcription-polymerase chain reaction. Positive signals for the presence of m2 mRNA were found in rat heart, brainstem, cerebral cortex, corpus striatum and hippocampus, which were used as positive controls. On the other hand, no expression of m2 was detected in lymphocytes. These results indicate that mRNA for the m2 subtype is absent in rat lymphocytes and that one or more other subtypes may be responsible for the reported results in binding experiments.

Identification of m3, m4 and m5 subtypes of muscarinic receptor mRNA in human blood mononuclear cells.

In this study we made use of the Reverse transcription-polymerase chain reaction to analyze the expression of mRNA for the five subtypes of muscarinic acetylcholine receptors in human blood mononuclear cells. mRNA for m3, m4 and m5 subtypes was detected, while mRNA for m1 and m2 muscarinic receptors was not found. Similar results were obtained for three different healthy human subjects studied. Interestingly, the m5 subtype was expressed at higher levels in blood mononuclear cells than in cerebral cortex. To our knowledge this is the first time that m5 muscarinic receptor mRNA has been found outside of the central nervous system.

Dendritic cell development from common myeloid progenitors.

Dendritic cells (DCs) are professional antigen-presenting cells which both initiate adaptive immune responses and control tolerance to self-antigens. It has been suggested that these different effects on responder cells depend on subsets of DCs arising from either myeloid or lymphoid hematopoietic origins. In this model, CD8 alpha+ Mac-1- DCs are supposed to be of lymphoid while CD8 alpha- Mac-1+ DCs are supposed to be of myeloid origin. Here we summarize our findings that both CD8 alpha+ and CD8 alpha- DCs can arise from clonogenic common myeloid progenitors (CMPs) in both thymus and spleen. Therefore CD8 alpha expression DCs does not indicate a lymphoid origin and differences among CD8 alpha+ and CD8 alpha- DCs might rather reflect maturation status than ontogeny. On the basis of transplantation studies, it seems likely that most of the DCs in secondary lymphoid organs and a substantial fraction of thymic DCs are myeloid-derived.

Lymphoid development from hematopoietic stem cells.

Mechanisms and pathways for commitment to the lymphoid lineage from hematopoietic stem cells (HSC) remain controversial. The interleukin-7 receptor (IL-7R) transduces nonredundant signals for both T- and B-cell development. Recently, we identified a clonogenic common lymphoid progenitor population in mouse bone marrow that can give rise to T, B, and natural killer (NK) cells, but lacks myeloid differentiation capacity. These cells are not self-renewing stem cells, but progenitors that have a limited life span. HSC do not express IL-7R, and the upregulation of the IL-7R occurs at the stage of common lymphoid progenitors. The IL-7R mediates nonredundant signals to reinforce the survival of developing T cells, and to promote rearrangement of immunoglobulin heavy chain genes in B-cell progenitors. Thus, common lymphoid progenitors exist in early hematopoiesis, and expression of the IL-7R is a critical step in the initiation of lymphoid development from HSC.

Lactic acid concentration in peritoneal fluid of normal and diseased horses.

Peritoneal fluid from each of 15 clinically healthy horses and five horses with acute abdominal disease was evaluated for lactic acid concentration. The normal range was 2-7--13-4 mg/dl. Simultaneous blood and peritoneal fluid samples from healthy horses revealed consistently lower lactic acid concentrations in the peritoneal fluid than in the blood, whereas peritoneal fluid lactic acid levels were consistently greater than blood levels in the diseased horses. The diseased horses had highly significant (P less than 0-005) increases in both blood and peritoneal fluid lactic acid concentrations compared with those in healthy horses.

Renal function of the pony and the horse.

Simultaneous renal clearances of inulin (CIN), p-aminohippurate (CPAH), and creatinine (CCR) were measured in hydrated mares (6 ponies and 2 horses). The CIN and CPAH were determined during steady-state infusion at 3 different infusion rates. A 6-fold change in plasma IN concentration did not produce alteration in CIN, nor was there a difference between the ponies and horses (P greater than 0.2). The overall average (mean +/- SEM) was 190.6 +/- 5.89 ml . min-1 . 100 kg of body weight-1. There was no difference noted between simultaneous CIN and CPAH. Clearance of PAH remained essentially constant during the change in plasma PAH from 0.33 mg/dl to 5.27 mg/dl. The extraction ratio of PAH for the nonanesthetized pony was 0.966. Effective renal plasma flow (CPAH) of the pony exceeded that of the horse.

Gentamicin toxic nephropathy in horses with disseminated bacterial infection.

Three clinical cases of toxic nephropathy in young horses were ascribed to gentamicin toxicity. Criteria for defining gentamicin-induced nephrotoxicosis were a serum urea nitrogen value greater than the pretreatment value or cylindruria, hematuria, and proteinuria in the absence of pyuria and bacteriuria. Recommended doses of gentamicin had been given in all cases. The nephropathy was reversible in 1 case in which the toxicosis was detected early and was treated by volume diuresis and drug withdrawal.

Epidural melanoma causing posterior paresis in a horse.

An aged gray stallion was examined because of fullminating posterior paresis, bladder paralysis, and perineal anesthesia. Lower motor neuron dysfunction was detected at the lumbosacral level of the spinal cord, and cerebrospinal fluid was yellow. After brief supportive treatment, the horse died. Necropsy revealed a single epidural melanoma at L5-6. The absence of cutaneous melanotic growth, absence of organ involvement, and extensive vertebral remodeling indicated the neoplasm to have been primary and to have been present for an extended period. Neurologic dysfunction was acute and progressive, as a result of spinal cord compression by the neoplasm.

Agammaglobulinemia in a horse.

Immunologic deficiency was suspected in an 18-month-old Standardbred horse with persistent fever, multifocal bacterial infection, and neutropenia with a large number of immature neutrophils. Serum protein electrophoresis revealed marked depression of the gamma-globulin fraction (0.2 g/100 ml). Immunologic testing and histologic examination of lymphoid tissues identified the immune deficit as agammaglobulinemia. Serum concentrations of immunoglobulin (Ig)G and IgG(T) were initially low and declined with time; IgM and IgA were not detectable. The horse failed to produce antibodies when inoculated with foreign antigens but had a positive cell-mediated skin reaction to intradermal phytolectin injection, and lymphocytes responded normally to in vitro stimulation by mitogens. Histologic examination of lymphoid tissues revealed absence of germinal centers and plasma cells.

Mice defective in two apoptosis pathways in the myeloid lineage develop acute myeloblastic leukemia.

Fas-deficient (Fas(lpr/lpr)) mice constitutively expressing Bcl-2 in myeloid cells by the hMRP8 promoter often develop a fatal disease analogous to human acute myeloblastic leukemia (AML-M2). Hematopoietic cells from leukemic Fas(lpr/lpr)hMRP8bcl-2 animals form clonogenic blast colonies in vitro and can transfer disease to wild-type mice. In vitro ligation of Fas on Fas+/+ hMRP8bcl-2 marrow cells depletes approximately 50% of myeloid progenitor activity, demonstrating that Bcl-2 can only partially block Fas-mediated death signals in myelomonocytic progenitors. In addition, Fas(lpr/lpr) marrow contains greatly increased numbers of myeloid colony-forming cells as compared to Fas+/+ controls. Taken together, these data suggest that Fas has a novel role in the regulation of myelopoiesis and that Fas may act as a tumor suppressor to control leukemogenic transformation in myeloid progenitor cells.

A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.

Haematopoietic stem cells give rise to progeny that progressively lose self-renewal capacity and become restricted to one lineage. The points at which haematopoietic stem cell-derived progenitors commit to each of the various lineages remain mostly unknown. We have identified a clonogenic common lymphoid progenitor that can differentiate into T, B and natural killer cells but not myeloid cells. Here we report the prospective identification, purification and characterization, using cell-surface markers and flow cytometry, of a complementary clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Common myeloid progenitors give rise to either megakaryocyte/erythrocyte or granulocyte/macrophage progenitors. Purified progenitors were used to provide a first-pass expression profile of various haematopoiesis-related genes. We propose that the common lymphoid progenitor and common myeloid progenitor populations reflect the earliest branch points between the lymphoid and myeloid lineages, and that the commitment of common myeloid progenitors to either the megakaryocyte/erythrocyte or the granulocyte/macrophage lineages are mutually exclusive events.

Expression of cholinergic muscarinic receptor subtypes mRNA in rat blood mononuclear cells.

The cholinergic system plays a role in the neuroimmune network. White blood cells have been found to express muscarinic and nicotinic cholinergic receptors as well as acetylcholinesterase on their surface, and cholinergic agents have been shown to modulate immune functions. Although the presence of muscarinic receptors on white blood cells has been well documented by binding and functional studies, the subtype(s) of muscarinic receptor present on these cells is still unknown. We have previously shown the absence of m2 muscarinic receptor subtype mRNA in rat mononuclear cells (Costa et al., 1994). The expression of m1, m3, m4 and m5 mAChR mRNA was analyzed in this study in rat peripheral blood mononuclear cells by Northern blotting hybridization experiments and reverse transcription-polymerase chain reaction. Only mRNA of the m3 subtype was detected in rat mononuclear cells, at levels about 100 times lower than in brain. Traces of m4 subtype mRNA were found in lymphocytes, at levels about 10,000 times lower than the cerebral cortex, while m5 mRNA was undetectable.