Widespread expression of SAA and Hp RNA in bovine tissues after evaluation of suitable reference genes.

The serum amyloid A (SAA) and haptoglobin (Hp) are the most prominent acute phase proteins (APPs) in cow. Liver mainly produces APPs, but extra hepatic expression has also been demonstrated in some tissues. The major aim of the present study was to assess the constitutive SAA and Hp mRNA expression by quantitative PCR (qPCR) in a wide panel of 33 bovine tissues, including gastrointestinal tract, respiratory system, urogenital system, mammary gland, hematopoietic system, central nervous system, eye, thyroid and heart. Normalization of gene expression in different samples requires reference genes, which are stably expressed. Therefore, seven reference genes were investigated (ACTB, GAPDH, HMBS, SDHA, YWHAZ, SF3A1, EEF1A2) and three genes, namely SF3A1, HMBS and ACTB, were selected after assessing their stability with geNorm™ and NormFinder(©) softwares. The qPCR analysis confirmed liver as the principal source of SAA and Hp, but also identified both APPs' mRNA in almost all tissues. The highest expression rate of SAA was found in thyroid, followed by pancreas and submandibulary gland. Hp mRNA expression was detected at high concentration in pancreas and submandibulary gland. The present data indicated a widespread expression of SAA and Hp also in non pathological conditions, thus envisaging a possible role as immunomodulatory and protective molecules. To understand where SAA and Hp come from is the prerequisite to their utilization as Acute Phase Reaction biomarkers.

Foe turned friend: multiple functional roles attributable to hyper-activating stem cell factor receptor mutant in regeneration of the haematopoietic cell compartment.

OBJECTIVES: Stem cell factor receptor, c-kit, is considered to be the master signalling molecule of haematopoietic stem cells. It develops the orchestral pattern of haematopoietic cell lineages, seen by its varying degree of omnipresence in progenitors, lineage committed and mature cells. We have investigated the effect of over-expressing c-kit on early recovery of the haematopoietic compartment, in irradiated hosts. MATERIALS AND METHODS: Normal bone marrow cells (BMCs) were transfected with Kit(wt) (wild-type c-kit) or its variant Kit(mu) (asp814tyr) by electroporation. Lethally irradiated mice were transplanted with normal or transfected congeneic BMCs. The effect of ectopic expression of c-kit on haematopoietic cell recovery was determined by analysing donor-derived cells. Furthermore, effects of both types of c-kit over-expression on progenitor and lineage-committed cells were examined by flow cytometric analysis of Sca-1 and lineage-committed (Lin(+)) cells respectively. RESULTS: Hyper-activating Kit(mu) significantly improved recovery of the haematopoietic system in irradiated hosts. In vivo results showed that the donor-derived c-kit(+) cell population was increased to more than 3-fold in the case of Kit(mu)-transfected cells compared to normal and Kit(wt) over-expressing BMCs. In general, survival of progenitor and committed cell was improved in the Kit(mu) over-expressing system compared to the other two cohorts. CONCLUSION: These results suggest that recruitment of the hyper-activating variant of c-kit (Kit(mu)) lead to early recovery of the bone marrow of lethally irradiated mice.

Defining APOBEC3 expression patterns in human tissues and hematopoietic cell subsets.

Human APOBEC3 enzymes are cellular DNA cytidine deaminases that inhibit and/or mutate a variety of retroviruses, retrotransposons, and DNA viruses. Here, we report a detailed examination of human APOBEC3 gene expression, focusing on APOBEC3G (A3G) and APOBEC3F (A3F), which are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) infection but are suppressed by HIV-1 Vif. A3G and A3F are expressed widely in hematopoietic cell populations, including T cells, B cells, and myeloid cells, as well as in tissues where mRNA levels broadly correlate with the lymphoid cell content (gonadal tissues are exceptions). By measuring mRNA copy numbers, we find that A3G mRNA is approximately 10-fold more abundant than A3F mRNA, implying that A3G is the more significant anti-HIV-1 factor in vivo. A3G and A3F levels also vary between donors, and these differences are sustained over 12 months. Responses to T-cell activation or cytokines reveal that A3G and A3F mRNA levels are induced approximately 10-fold in macrophages and dendritic cells (DCs) by alpha interferon (IFN-alpha) and approximately 4-fold in naïve CD4(+) T cells. However, immunoblotting revealed that A3G protein levels are induced by IFN-alpha in macrophages and DCs but not in T cells. In contrast, T-cell activation and IFN-gamma had a minimal impact on A3G or A3F expression. Finally, we noted that A3A mRNA expression and protein expression are exquisitely sensitive to IFN-alpha induction in CD4(+) T cells, macrophages, and DCs but not to T-cell activation or other cytokines. Given that A3A does not affect HIV-1 infection, these observations imply that this protein may participate in early antiviral innate immune responses.

Phosphodiesterase 4 inhibitors augment levels of glucocorticoid receptor in B cell chronic lymphocytic leukemia but not in normal circulating hematopoietic cells.

Type 4 cyclic AMP (cAMP) phosphodiesterase (PDE4) inhibitors, a class of compounds in clinical development that activate cAMP-mediated signaling by inhibiting cAMP catabolism, offer a feasible means by which to potentiate glucocorticoid-mediated apoptosis in lymphoid malignancies such as B-cell chronic lymphocytic leukemia (B-CLL). In this study, we show that PDE4 inhibitors up-regulate glucocorticoid receptor (GRalpha) transcript levels in B-CLL cells but not T-CLL cells or Sezary cells or normal circulating T cells, B cells, monocytes, or neutrophils. Because GRalpha transcript half-life does not vary in CLL cells treated with the prototypic PDE4 inhibitor rolipram, the 4-fold increase in GRalpha mRNA levels observed within 4 h of rolipram treatment seems to result from an increase in GRalpha transcription. Rolipram treatment increases levels of transcripts derived from the 1A3 promoter to a greater extent than the 1B promoter. Treatment of B-CLL cells with two other PDE4 inhibitors currently in clinical development also augments GR transcript levels and glucocorticoid-mediated apoptosis. Washout studies show that simultaneous treatment with both drug classes irreversibly augments apoptosis over the same time frame that GR up-regulation occurs. Although treatment of B-CLL cells with glucocorticoids reduces basal GRalpha transcript levels in a dose-related manner, cotreatment with rolipram maintained GRalpha transcript levels above baseline. Our results suggest that as a result of their unusual sensitivity to PDE4 inhibitor-mediated up-regulation of GRalpha expression, treatment of B-CLL patients with combined PDE4 inhibitor/glucocorticoid therapy may be of therapeutic benefit in this disease.

Expression profiling of peroxisome proliferator-activated receptor-delta (PPAR-delta) in mouse tissues using tissue microarray.

Peroxisome proliferator-activated receptor-delta (PPAR-delta) is known as a transcription factor involved in the regulation of fatty acid oxidation and mitochondrial biogenesis in several tissues, such as skeletal muscle, liver and adipose tissues. In this study, to elucidate systemic physiological functions of PPAR-delta, we examined the tissue distribution and localization of PPAR-delta in adult mouse tissues using tissue microarray (TMA)-based immunohistochemistry. PPAR-delta positive signals were observed on variety of tissues/cells in multiple systems including cardiovascular, urinary, respiratory, digestive, endocrine, nervous, hematopoietic, immune, musculoskeletal, sensory and reproductive organ systems. In these organs, PPAR-delta immunoreactivity was generally localized on the nucleus, although cytoplasmic localization was observed on several cell types including neurons in the nervous system and cells of the islet of Langerhans. These expression profiling data implicate various physiological roles of PPAR-delta in multiple organ systems. TMA-based immunohistochemistry enables to profile comprehensive protein localization and distribution in a high-throughput manner.

Amelogenin, a major structural protein in mineralizing enamel, is also expressed in soft tissues: brain and cells of the hematopoietic system.

The amelogenin protein is considered as the major molecular marker of developing and mineralizing ectodermal enamel. It regulates the shape, size, and direction of growth of the enamel mineral crystallite. Recent data suggest other roles for amelogenin beyond regulation of enamel mineral crystal growth. The present study describes our recent discovery of amelogenin expression in soft tissues: in brain and in cells of the hematopoietic system, such as macrophages, megakaryocytes and in some of the hematopoietic stem cells. Reverse transcription-polymerase chain reaction (RT-PCR) followed by cDNA sequencing revealed, in mouse brain, two amelogenin mRNA isoforms: the full-length amelogenin including exon 4, and the isoform lacking exon 4. Immunohistochemistry revealed amelogenin expression in brain glial cells. Mouse macrophages were found to express the full-length amelogenin sequence lacking exon 4. Confocal microscopy revealed colocalization of amelogenin and CD41 (a megakaryocyte marker), as well as amelogenin and CD34 (a hematopoietic stem cell marker) in some of the bone marrow cells. The expression of amelogenin, a major structural protein of the mineralizing extracellular enamel matrix, also in cells of non-mineralizing soft tissues, suggests that amelogenin is multifunctional. Several different potential functions of amelogenin are discussed.

Expression of the c-ErbB-2/HER2 proto-oncogene in normal hematopoietic cells.

The HER2/c-ErbB-2 proto-oncogene is overexpressed in 25-30% of human breast cancers. We previously reported the c-ErbB-2 transcript in mononuclear cells (MNC) from bone marrow (BM), peripheral blood (PB), and mobilized PB (MPB). Here, we describe extensively the expression pattern of c-ErbB-2 mRNA and protein in normal adult hematopoietic tissue and cord blood (CB)-derived cells. Quantitative reverse transcriptase-polymerase chain reaction shows that the c-ErbB-2 transcript is expressed in hematopoietic cells at low levels if compared with normal epithelial and breast cancer cells. The c-ErbB-2 protein was detected predominantly in MNC from PB and CB by Western blot analysis. Flow cytometry revealed that CD15+, CD14+, and glycophorin A+ subpopulations express c-ErbB-2 protein, whereas lymphocytes are c-ErbB-2-negative. The c-ErbB-2 expression is higher in CB MNC. More than 90% of BM- and MPB-derived CD34+ progenitors are c-ErbB-2-negative; by contrast, 5-40% of CB-derived CD34+ progenitors express c-ErbB-2. We found that c-ErbB-2 protein is up-regulated during cell-cycle recruitment of progenitor cells. Similarly, it increases in mature, hematopoietic proliferating cells. This study reports the first evidence that the c-ErbB-2 receptor is correlated to the proliferating state of hematopoietic cells. Studies in progress aim to clarify the role of c-ErbB-2 in regulation of this process in hematopoietic tissues.

Clnk, a novel SLP-76-related adaptor molecule expressed in cytokine-stimulated hemopoietic cells.

We have identified a novel Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76)-related molecule which we have termed Clnk (for cytokine-dependent hemopoietic cell linker). Unlike its relatives SLP-76 and B cell linker protein (Blnk), Clnk is not expressed uniformly within a given hemopoietic cell lineage. Even though it can be detected in several cell types, including T cells, natural killer cells, and mast cells, its expression seems to be strictly dependent on sustained exposure to cytokines such as interleukin (IL)-2 and IL-3. Strong support for the notion that Clnk is involved in immunoreceptor signaling was provided by the observation that it inducibly associated with at least one tyrosine-phosphorylated polypeptide (p92) in response to immunoreceptor stimulation. Moreover, transient expression of Clnk caused an increase in immunoreceptor-mediated signaling events in a T cell line. Taken together, these results show that Clnk is a novel member of the SLP-76 family selectively expressed in cytokine-stimulated hemopoietic cells. Furthermore, they suggest that Clnk may be involved in a cross-talk mechanism between cytokine receptor and immunoreceptor signaling.

Correlation of T1 and T2 relaxation rates in normal bone-marrow water with serum ferritin concentration.

PURPOSE: This study was made to clarify the paramagnetic effect of iron stored in the hematopoietic tissue of bone marrow. MATERIAL AND METHODS: The T1 and T2 relaxation times of bone-marrow water in the L1-3 vertebrae of 20 healthy individuals were measured by MR imaging with a 1.5 T magnet. The chemical shift misregistration effect was used to isolate the bone-marrow water. The results were compared with the serum ferritin concentration. RESULTS AND CONCLUSION: Although no correlation between the T1 relaxation rate of the water fraction and the serum ferritin concentration was evident, the T2 relaxation rate of the water fraction showed strong linear correlation with the serum ferritin concentration (r = 0.87, p < 0.0001). Thus, T2 of bone-marrow water accurately reflects the amount of iron in normal bone marrow. This finding may be useful in the evaluation of the characteristics of hematopoietic tissue in bone marrow.

Detection of 8-hydroxydeoxyguanosine in K562 human hematopoietic cells by high-performance capillary electrophoresis.

A method for the detection of 8-hydroxydeoxyguanosine by high-performance capillary electrophoresis (HPCE) was developed. Separations were performed in an uncoated silica capillary (44 cm x 75 microns i.d.) with a P/ACE system with diode-array detector. The separation of purine deoxynucleosides and 8-hydroxydeoxyguanosine was optimized with regard to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.5), 25 degrees C, 25 kV, 20 s load and detection at 254 nm. This method allowed the detection of 8-hydroxydeoxyguanosine in the presence of a 10(5)-fold higher amount of deoxyguanosine. Isolated nuclei from K562 human hematopoietic cells were treated with 15 mM hydrogen peroxide for 2 h. The nuclei were extensively dialyzed and DNA was isolated, enzymatically hydrolyzed to the deoxynucleosides and analyzed by HPCE. DNA from hydrogen peroxide treated nuclei had a 4-fold higher content of 8-hydroxydeoxyguanosine than untreated controls. HPCE analysis of 8-hydroxydeoxyguanosine is fast and simple. Furthermore, it requires a very small sample volume, which makes it useful for biomedical and clinical applications.

Epidemiological characterization of newly recognized rat parvovirus, "rat orphan parvovirus".

Newly recognized rat parvovirus (rat orphan parvovirus: ROPV) was examined for viral excretion and persistence in infected rats, and also for infectivity to mice and hamsters. The virus appeared to replicate mainly in lymphoid or hematopoietic tissues, and was detected in feces, urine and oropharynx of the infected rats at 1 to 4 weeks postinfection. The infective virus was also detected in peripheral leukocytes and various tissues at an acute phase of infection, and decreased in every tissue at 8 weeks postinfection. Viral DNA, however, was persistent in lymphoid tissues at least up to 24 weeks postinfection. When the virus was inoculated to mice and hamsters, no evidence of viral production and antibody response was demonstrated. ROPV is assumed to be a variant of the known rat parvovirus which resulted to alter cell tropism and persist in lymphoid or hematopoietic tissues, in order to escape from host immune system.

Cell type- and differentiation stage-dependent expression of PML domains in rat, detected by monoclonal antibody HIS55.

Using mouse monoclonal antibody (mAb) HIS55, we identified a nuclear antigen (ag) that exhibited a staining pattern of discrete foci. Such foci could be detected in cells of many mammalian species. These nuclear foci were not associated with nuclear membrane, nucleoli, or mitotic chromosomes. In isolated rat liver nuclei, HeLa cells, and normal human and rat lymph nodes, staining of HIS55 colocalized with that of 5E10. 5E10 recognizes PML nuclear domains, multimolecular complexes of unknown function containing the product of PML gene and at least two other components. HIS55 foci were expressed widely in many tissues but the expression level varied in a cell type-specific manner, with the number of HIS55 nuclear foci ranging from 0 (as in neurons) to over 100 (as in megakaryocytes) and the size ranging from fine (as in cortical thymocytes) to very large (as in urethra epithelium). HIS55 ag expression level also varied among cells of the same lineage, as observed in embryonic development of rat and in the hemopoietic system of adult rat. The expression level of HIS55 foci roughly correlated with the overall rate of protein synthesis of cells, supporting a role of PML domains as transcription regulatory units. The expression of HIS55 foci, however, did not correlate with the growth index of cell populations. Our observations on normal tissues agreed with the hypothesis that PML domain expression is regulated by external, possibly site-dependent factors. We further supported this by demonstrating that PML domains in rat ventral prostate epithelia were upregulated upon castration.

The HEM proteins: a novel family of tissue-specific transmembrane proteins expressed from invertebrates through mammals with an essential function in oogenesis.

We report the identification of a new family of proteins, termed the HEM family, which show distinct expression patterns in blood cells and the central nervous system. Through the isolation and characterization of the corresponding brain-specific Drosophila (dhem-2) and rat orthologues (Hem-2), and through the detection of the Caenorhabditis elegans Hem-2 orthologue in the database, we show that this family is conserved throughout evolution. HEM proteins show a conserved length ranging from 1118 to 1126 amino acid residues. Moreover, they are at least 35% identical with each other and harbour several conserved membrane-spanning domains, indicative for their location on the cell surface. One of the members, the Drosophila orthologue dhem-2, was analysed in detail for its spatial expression pattern during development and for its mutant phenotype. dhem-2 is expressed maternally in the oocyte and shows uniform expression during the first half of embryogenesis, but becomes restricted to the brain and the nervous system during late embryogenesis, consistent with the expression of its vertebrate orthologue in the brain. One P-element insertion, located 39 base-pairs downstream from the dhem-2 transcription start site, causes female sterility, due to the fact that developmental processes in the oocyte are disturbed. Of the vertebrate HEM family members, the mammalian Hem-1 gene is expressed only in cells of hematopoietic origin, while Hem-2 is preferentially expressed in brain, heart, liver and testis.

Immunohistochemical analysis of Mcl-1 protein in human tissues. Differential regulation of Mcl-1 and Bcl-2 protein production suggests a unique role for Mcl-1 in control of programmed cell death in vivo.

The mcl-1 gene encodes an approximately 37-kd protein that has significant homology with Bcl-2, an inhibitor of programmed cell death that is expressed in many types of long-lived cells. In this study we determined the in vivo patterns of Mcl-1 protein production in normal human tissues by immunohistochemical means, using specific polyclonal antisera, and made comparisons with Bcl-2. Like Bcl-2, Mcl-1 immunostaining was observed in epithelial cells in a variety of tissues, including prostate, breast, endometrium, epidermis, stomach, intestine, colon, and respiratory tract. However, often the expression of mcl-1 and bcl-2 in complex epithelia occurred in gradients with opposing directions, such that Bcl-2 immunostaining tended to be higher in the less differentiated cells lining the basement membrane, whereas Mcl-1 immunostaining was more intense in the differentiated cells located in the upper layers of these epithelia. The in vivo patterns of mcl-1 and bcl-2 expression were also strikingly different in several other tissues as well. Within the secondary follicles of lymph nodes and tonsils, for example, germinal center lymphocytes were Mcl-1 positive but mostly lacked Bcl-2; whereas mantle zone lymphocytes expressed bcl-2 but not mcl-1. Intense Mcl-1 immunoreactivity was also detected in several types of neuroendocrine cells, including the adrenal cortical cells that are Bcl-2 negative, sympathetic neurons that also contain Bcl-2, a subpopulation of cells in the pancreatic islets, Leydig cells of the testis, and granulosa lutein cells of the ovarian corpus luteum but not in thyroid epithelium, which is strongly Bcl-2 positive. Little or no Mcl-1 was detected in neurons in the brain and spinal cord, in contrast to Bcl-2, which is present in several types of central nervous system neurons. Conversely, strong Mcl-1 immunostaining was found in cardiac and skeletal muscle, which contain comparatively less Bcl-2. Additional types of cells that are Bcl-2-negative but that expressed mcl-1 include chondrocytes and hepatocytes. These findings demonstrate that mcl-1 expression is widespread in vivo and imply that the Mcl-1 and Bcl-2 proteins fulfill different roles in the overall physiology of cell death regulation.

Tissue distribution of liver regulating protein. Evidence for a cell recognition signal common to liver, pancreas, gonads, and hemopoietic tissues.

Liver regulating protein (LRP) is an integral plasma membrane protein that plays a critical role in maintaining the differentiated phenotype of adult rat hepatocytes by mediating cell-cell interactions with rat liver epithelial cells. Using a specific monoclonal antibody (MAb L8) capable of inhibiting the interactions between these two cell types, the cellular distribution of LRP was analyzed in the liver. Various cell types, including hepatocytes and several sinusoidal cells, were found to be positive, whereas vascular endothelial cells and bile duct cells were consistently negative. This observation led us to question whether cells of nonhepatic origin would also express LRP. We show that MAb L8 immunoreactive material was detected in only three groups of tissues and corresponded to molecules similar to LRP but with different molecular weights. LRP-like molecules were demonstrated on acinar cells of the exocrine pancreas and on all hemopoietic cells regardless of their localization in the organism. LRP-like molecules were also expressed by germ cells and surrounding feeder cells in the testis and ovary in a stage-dependent manner. These results demonstrate the existence of a family of LRP proteins and strongly suggest a critical role for these molecules in regulating cell-cell communication in specific tissues.

Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1.

The ETS oncogene family member PU.1 is a transcriptional activator that is dysregulated by Friend erythroleukemia virus insertion. Northern analysis found that PU.1 is highly expressed in cells of myeloid and B-lymphoid origin, but not expressed at all in a number of nonhematopoietic tissues. Interferon-gamma and retinoic acid downregulated PU.1 expression in marrow macrophages. In situ immunohistochemistry found that PU.1 is expressed only in early granulocytic and erythroid cells and megakaryocytes, but not in mature erythroid cells, mature granulocytes, endothelial cells, or osteocytes. Thus, its expression pattern makes PU.1 a candidate for a genetic determinant of lineage commitment and stage progression in blood cell development. It also lends insight into how PU.1 might play a role in Friend virus erythroleukemia.

Patterns of X chromosome inactivation in haematopoietic cells of female carriers of X linked severe combined immunodeficiency.

We evaluated the use of methylation analysis at the DXS255 locus as a method for carrier detection in X-linked severe combined immunodeficiency (XSCID). We also investigated the variations of X inactivation patterns in several haematopoietic cell lineages of XSCID carriers, both within and between XSCID pedigrees.

A novel transcript overlapping the myelin basic protein gene.

Myelin basic protein (MBP) is a major constituent of myelin synthesized by oligodendrocytes and Schwann cells. We have investigated the expression of mouse MBP RNAs outside the nervous system. Nuclease protection experiments indicate that RNAs containing exon 1 and not the six downstream exons of the MBP gene are transcribed in various hemopoietic tissues. We have isolated a hemopoietic MBP-related (HMBPR) cDNA clone from a mouse bone marrow cDNA library screened with an MBP cDNA probe. This clone contains exons 1a and 1b and a part of intron 1 of the MBP gene. An additional 5' region, encoded by at least three unidentified exons, lies upstream of exon 1a. The HMBPR clone corresponds to a 5-kb RNA expressed in bone marrow, spleen, thymus, and macrophagic cells. This transcript is expressed at a similar level in brain, although at a lower level than the classical 2-kb mRNA. These data indicate that a new transcript, overlapping the MBP transcription unit and controlled by a distinct promoter, is expressed in hemopoietic tissues. This RNA might encode a 21-kDa protein sharing a common domain with MBP.

Immunological detection of the oestradiol receptor protein in cell lines derived from the lymphatic system and the haematopoietic system: variability of specific hormone binding in vitro.

Extracts of human MCF 7 mammary carcinoma cells, the human lymphoblastoid cell lines AEH 1 and IM 9, T-cell derived CCRF cells, HL 60 myeloic leukaemia cells and murine myeloma cells SP 0 and NS I were analysed for immunoreactivity with polyclonal goat antibodies raised against homogeneous preparations of C-terminal fragments (32 kDa) of porcine uterine oestradiol receptor (ER). Whole cells and low speed cytosols were analysed for specific oestradiol-binding activity. ERs were enriched from cell extracts by either fractionated ethanol precipitation (0-25% (v/v) ethanol) and/or microscale-immunoaffinity chromatography. Immunoreactive proteins of identical molecular weight (approximately 65 kDa) were detected in all cell lines examined. Whole cell binding assays showed specific oestradiol-binding activity in MCF 7, IM 9 and CCRF cells. Borderline binding was found in HL 60 myeloid cells. No specific binding could be detected in AEH 1, NS I and SP 0 cells. Identical results were obtained using agar-electrophoresis after dextran-coated charcoal treatment. Immunoaffinity purified ERs from MCF 7, AEH 1 and HL 60 cells were subjected to limited proteolysis, where identical tryptic fragments were generated. In conclusion, we have confirmed by immunological methods that ERs are expressed in a variety of cell lines derived from the immune system and the haematopoietic system. The lack of specific hormone binding or very low-affinity hormone binding in some of the cells examined may be due to post-translational events or point mutations.