Bovine hepatic and adipose retinol-binding protein gene expression and relationship with tumor necrosis factor-α.

Retinol-binding protein (RBP) is the main transport system for retinol in circulation, is a relatively small protein with one binding site for retinol in the all-trans form, and is bound to transthyretin. The objectives of this study were to characterize the temporal pattern of bovine hepatic mRNA expression of RBP during the periparturient period and to determine if a relationship exists between the expression of RBP and that of tumor necrosis factor (TNF)-α in dairy cows. In experiment 1, we assessed hepatic mRNA expression of RBP during the periparturient period. Liver tissues were sampled from periparturient dairy cows (n=9) at -21, -4, +1, +7, and +21 relative to parturition and frozen in liquid N(2). Total RNA was extracted from each tissue sample and cDNA was generated. Gene expressions of RBP and ß-actin (as a housekeeping gene) were measured as relative quantity using reverse transcription-PCR. Data were analyzed using cycle threshold values, adjusted to ß-actin, and significance was determined at P<0.05. Serum samples (-21, -4, +1, +7, and +21 relative to parturition) were analyzed for retinol concentration using a standard HPLC-based method. Cows had variable expression of hepatic RBP and serum retinol over the transition period, with a decline near parturition and a rebound toward prepartum levels later in lactation. In experiment 2, liver and visceral (intestinal) adipose tissues were sampled from dairy cows (n=28) at slaughter. Expression of RBP and TNF-α was detected in all samples and variations among cows were highly significant for both genes. Across tissues, expression of RBP was positively correlated with that of TNF-α (r=0.60). Within adipose tissue, expression of RBP and TNF-α was weakly correlated (r=0.23), whereas in hepatic tissue, expression was strongly correlated (r=0.62). In experiment 3, late-lactation dairy Holstein cows were blocked by parity and feed intake, and randomly assigned to control, recombinant bovine (rb)TNF challenge, or pair-fed control treatment (n=5/treatment). Cows were injected with either rbTNF (subcutaneous injection of 2 µg/kg of body weight in saline) or sterile saline (control and pair-fed control animals) once daily for 7d. Liver biopsy was performed on d 7 and samples were processed for expression of RBP and TNF-α. Although TNF challenge caused an upregulation of hepatic TNF-α expression, as expected, it did not alter hepatic RBP expression. Overall, the temporal pattern of hepatic RBP gene expression during the periparturient period followed, to a great extent, that of plasma retinol. Although a strong positive correlation was previously detected between bovine hepatic RBP and TNF-α transcripts, rbTNF challenge did not cause alter RBP expression. These observations collectively imply that regulation of RBP at the transcription level is influenced by physiological state but may be independent from that of transthyretin, which is altered by proinflammatory stimuli (such as TNF-α) via induction of transcription factor nuclear factor-interleukin 6.

Human milk oligosaccharides promote the growth of staphylococci.

Human milk oligosaccharides (HMO), which constitute a major component of human milk, promote the growth of particular bacterial species in the infant's gastrointestinal tract. We hypothesized that HMO also interact with the bacterial communities present in human milk. To test this hypothesis, two experiments were conducted. First, milk samples were collected from healthy women (n = 16); culture-independent analysis of the bacterial communities was performed, HMO content was analyzed, and the relation between these factors was investigated. A positive correlation was observed between the relative abundance of Staphylococcus and total HMO content (r = 0.66). In a follow-up study, we conducted a series of in vitro growth curve experiments utilizing Staphylococcus aureus or Staphylococcus epidermidis and HMO isolated from human milk. HMO exhibited stimulatory effects on bacterial growth under various nutritional conditions. Analysis of culture supernatants from these experiments revealed that HMO did not measurably disappear from the culture medium, indicating that the growth-enhancing effects were not a result of bacterial metabolism of the HMO. Instead, stimulation of growth caused greater utilization of amino acids in minimal medium. Collectively, the data provide evidence that HMO may promote the growth of Staphylococcus species in the lactating mammary gland.

Transient and persistent expansions of large granular lymphocytes (LGL) and NK-associated (NKa) cells: the Yorkshire Leukaemia Group Study.

A survey of 870 different adult blood samples (primarily from patients with non-haematological disorders) found that 269 (31%) had increased proportions (> 25%) and/or absolute numbers (> 1.0 x 10(9)/l) of morphologically-defined large granular lymphocytes (LGL), and/or phenotypically-defined NK-associated (NKa) cells. Of these, 112 were re-analysed at least 6 months after initial presentation and were classified as 'persistent' (92/112) or 'transient' (20/112) according to whether or not the original abnormality was still present. Lymphocyte counts in most patients with persistent abnormalities were within normal limits (18/92) or slightly increased (68/92), with only six having a lymphocytosis exceeding 10.0 x 10(9)/l. With the exception of five persistent LGL expansions in which the granular lymphocytes did not express NKa determinants (designated LGL+NKa-), the remaining 87 cases could be phenotypically grouped according to their primary abnormality as CD8+NKa+ (n = 33), CD4+ NKa+ (n = 14), CD8dim+NKa+ (n = 7) or CD8-NKa+ (n = 33). TCR genotypic studies in 58 patients showed that the 16 patients with rearranged TCR components were restricted to the CD8+NKa+ group and that, in most of these, the CD8+ fraction showed abnormal relative CD16/CD56 expression. Persistent neutropenia (n = 15) also appeared to be associated with primary abnormalities of CD8+NKa+ cells (12/15), with 10 of these additionally showing rearranged TCR genes. In contrast, persistently increased CD8dim+NKa+ and CD8-NKa+ components did not appear to phenotypically differ from their corresponding 'counterparts' in normal bloods or in patients with transient LGL/NKa+ abnormalities. This survey has therefore established that persistent LGL/NKa+ abnormalities are considerably more common than suggested in published work, that a high proportion of patients with expanded CD8+NKa+ components, with quite diverse clinical histories, show evidence of clonal lymphoid populations, and that the clonal nature of such disorders appears to be associated with abnormal NKa phenotypic patterns.

Patterns of CD16 and CD56 expression in persistent expansions of CD3+NKa+ lymphocytes are predictive for clonal T-cell receptor gene rearrangements. The Yorkshire Leukaemia Group.

Phenotypic characteristics, and correlations between the expression of membrane NK-associated (NKa) determinants (CD11b, CD16, CD56 and CD57) and T cell receptor (TCR) genotypic patterns, were examined in 25 patients with persistent (greater than 6 months) expansions of CD3+WT31+NKa+ (CD8+ and CD8dim+) lymphocytes. These studies showed that distinct NKa phenotypic profiles were restricted to cases with rearranged TCR configurations and that clonal CD3+NKa+ components could be predicted in most cases by assessing relationships between membrane CD16 and CD56 expression. For all normal NKa subpopulations, there was a high correlation (P less than 0.0001; n = 31) between the expression of these two membrane determinants. Markedly increased CD16 expression by CD3+NKa+ cells, in relation to CD56 (i.e. a high CD16:CD56 ratio), was found exclusively in cases with rearranged TCR (13/16 cases); 2/3 of the remaining cases showing significantly reduced CD16:CD56 ratios and high (greater than 2.0) CD3+CD56+ absolute numbers. In contrast, 7/9 of the germline TCR cases had a normal CD16:CD56 ratio and 2/9 a decreased ratio with low (less than 1.0) CD3+CD56+ absolute numbers. A high ratio of CD16:CD56 expression by CD3+NKa+ lymphocytes was therefore informative for 82% of TCR rearrangements in this series; and analysis of CD16 and CD56 expression was predictive for germline and rearranged TCR configurations in 24/25 persistent CD3+NKa+ expansions.

Polycythaemia and neuroblastoma.

The treatment of primary proliferative polycythaemia (polycythaemia rubra vera) may include radioactive phosphorus (P32) in conjunction with venesection. Acute leukaemia or carcinoma can be associated with the use of P32. We present a case of primary proliferative polycythaemia treated by repeat venesection together with P32 whose follow-up was complicated by the development of malignant neuroblastoma.

Proliferative Hairy Cell Leukaemia (HCL-v) Resistant to Alpha-Interferon: Clinical, Diagnostic and In-Vitro Cellular Characteristics.

This report details the clinical and cellular features of a patient with 'proliferative' hairy cell leukaemia (HCL-v) who presented with a WBC count of 345 x 10(9)/1. In contrast to typical HCL, there was no significant clinical response to splenectomy or treatment with alpha-interferon but leucophoresis was found to be important in maintaining a stable WBC count. Additionally, morphological (light and electron microscopy), cytochemical (ANAE +, AcP +, TRAcP -) and immunophenotypic (CDlc +, CDllc +, CD19 +, CD20 +, CD23-, CD25-, FMC7 +, HC2-) studies of HCL-v cells were undertaken together with an extensive analysis of cellular characteristics (including RNA/DNA synthesis) following in-vitro culture. In contrast to typical HCL cells, but in common with CLL/PLL cells, TPA did not induce the 'acquisition' of long fibroblast-like cytoplasmic extensions. However, plasmacytoid features could be induced by culture in the presence of TPA plus the calcium ionophore A23187. Furthermore, although some TRAcP isoenzyme synthesis could be induced by TPA, its level of staining (IEF) did not reach the same intensity as that usually observed for HCL. In-vitro culture studies, with TPA and TPA/A23187 as well as a number of immunomodulators, were also used to examine the rates of immunoglobulin (Ig) and beta(2)-microglobulin (B(2)m) secretion. TPA alone was the most potent stimulator of both Ig and B(2)m production whereas the addition of A23187 to TPA cultures induced a marked suppression of Ig production but only minimally affected B(2)m secretion. For comparison, bryostatin-1, r IFN-lambda, r IL-2 and r TNF were all considerably less effective than TPA in the induction of B(2)m production. These observations further support the contention that HCL-v represents a leukaemia subtype intermediate between HCL and B-PLL and that recognition of this variant is of particular importance in therapeutic management.

Factors affecting fetal survival after intrauterine transfusion for rhesus isoimmunization.

Intrauterine transfusion of the fetus is described in 165 pregnancies. The overall fetal survival rose from 28 per cent in 1964 to 1969 to 42-5 per cent in 1973 to 1975. Apart from technical complications of the procedure itself, the factors most likely to affect fetal survival were the gestational age and amniotic fluid optical density difference before the first intrauterine transfusion, the birth weight and the cord blood haemoglobin level.