Modification of protein transfer across blood/cerebrospinal fluid barrier in response to altered plasma protein composition during development.

A developmentally regulated protein-specific transfer mechanism across choroid plexus epithelial cells has previously been proposed to contribute to the characteristically high concentration of protein in cerebrospinal fluid (CSF) in the immature brain. Here we demonstrate that this mechanism is sensitive to protein variations in plasma resulting in changed numbers of transferring cells for individual proteins and altered transfer into the CSF. Pups of Monodelphis domestica at postnatal day (P)9, P65 and P110 were injected intraperitoneally with either adult Monodelphis plasma or exogenous bovine fetuin. Samples of CSF, blood and brain were collected from terminally anaesthetized animals 3-48 h later. The concentration of total protein was measured and levels of albumin, hemopexin, α-fetoprotein and bovine fetuin were estimated by western blotting. Numbers of lateral ventricular choroid plexus cells positive for total and individual plasma proteins were counted in paraffin sections of brains stained with appropriate antibodies. Following intraperitoneal injections, the content of proteins in the CSF increased at all three ages, but the concentration increased only in the CSF of older animals. The total numbers of plexus cells positive for plasma protein did not change significantly, but cells positive for individual proteins did. Fetuin was detected in all protein-positive cells, but apparently displaced α-fetoprotein and, to a lesser degree, hemopexin. The results indicate that protein transfer across the blood/CSF barrier appears to be regulated by a molecular recognition mechanism that is probably saturable but may not be as specific for individual proteins as previously suggested.

Effect of minocycline on inflammation-induced damage to the blood-brain barrier and white matter during development.

Damage to white matter in some premature infants exposed to intrauterine infections is thought to involve disruption of the blood-brain barrier. We have examined the effect of minocycline, an agent reported to reduce brain damage resulting from inflammation, on inflammation-induced disruption of the blood-brain barrier and damage to white matter. Post-natal marsupial opossums (Monodelphis domestica) were studied as most brain development in this species occurs after birth. Single intraperitoneal lipopolysaccharide (LPS) injection (0.2 mg/kg) with or without minocycline (45 mg/kg) at post-natal day (P)35 caused short-lasting barrier breakdown to plasma proteins but not to (14)C-sucrose. By P44, blood-brain barrier integrity was intact but a reduced volume of white matter was present. At P44 after prolonged inflammation (5 x 0.2 mg/kg LPS at 48 h intervals), proteins from blood were observed within brain white matter and permeability to (14)C-sucrose in the hindbrain increased by 31%. The volume of the external capsule and the proportion of myelin were 70 and 57%, respectively, of those in control animals. Minocycline administered during prolonged inflammation restored blood-brain barrier integrity but not LPS-induced damage to white matter. These data suggest that long-term changes in blood-brain barrier permeability occur only after a prolonged period of inflammation during development; however, damage to white matter can result from even a short-lasting breakdown of the barrier. Manipulation of the inflammatory response may have implications for prevention of some developmentally induced neurological conditions.

Propranolol-induced postoperative hypertension following coronary artery bypass grafting.

Fifteen patients receiving propranolol preoperatively and undergoing coronary artery bypass grafting had serum propranolol levels determined preoperatively and at several times early postoperatively. In addition, the patients' hemodynamic parameters and postoperative sodium nitroprusside dose requirements were monitored. All patients had significant multivessel disease and normal left ventricular function. Preoperative serum propranolol levels ranged from 16 to 243 ng/ml, with a mean level of 92 +/- 17 ng/ml; propranolol measured at the end of bypass ranged from 0 to 92 ng/ml, with a mean level of 23 +/- 7 ng/ml. Fourteen patients (93%) had hypertension postoperatively and required intravenous sodium nitroprusside to maintain mean blood pressure at or below 90 mm Hg. According to linear regression analysis, the severity of the postoperative hypertension or, specifically, the nitroprusside dose requirements, correlated significantly with the patients' serum propranolol levels postoperatively (correlation coefficient, R = 0.76, with p less than 0.001). The one normotensive patient had no detectable serum propranolol at any time postoperatively. No correlation was noted between the patient's preoperative serum propranolol levels and the need for nitroprusside therapy postoperatively. These results demonstrate that there is a significant relationship between residual propranolol and the development of hypertension postoperatively.

Application of fluorescence in situ hybridisation to chromosome analysis of aged bone marrow smears.

AIMS: To evaluate the reliability of fluorescence in situ hybridisation (FISH) in the retrospective cytogenetic assessment of old bone marrow smears stored for periods of up to 20 years. METHODS: A series of bone marrow smears either Romanowsky stained, or frozen and unstained, and aged from one month to 20 years were hybridised with biotin labelled probes specific for the centromeric regions of human chromosomes X, 6, and 18. Sites of hybridisation were detected with fluoresceinated avidin. One hundred to 400 cells from each preparation were examined and the number of signals observed was recorded. RESULTS: All smears exhibited signals in most cells examined. In cytogenetically normal cases, an average 67.6% of cells (range 36%-90%) demonstrated the appropriate number of X centromere signals. In those samples known to contain extra chromosomes X, 6, or 18 the presence of cells with the abnormal copy number was clearly detected in each case. CONCLUSION: When applied in the way described, FISH can give consistent and accurate results with a variety of archival bone marrow smears, including aged prestained material. This will permit retrospective assessment of specific cytogenetic abnormalities in patients with leukaemia using their initial diagnostic slides even where these are several years old.

Chromosomes in childhood acute lymphoblastic leukaemia: karyotypic patterns in disease subtypes.

To define further the clinical importance of cytogenetic analysis in acute lymphoblastic leukaemia (ALL) a prospective study was performed on 139 unselected children. Analyses were considered adequate in 104, of whom 35 were normal and 69 had clonal abnormalities. Abnormalities were categorised according to banded chromosome analysis as well as chromosome count. Karyotypes were correlated with clinical and laboratory features at diagnosis and with survival. Of the successful analyses, thirty five (34%) children had no abnormalities; this group contained an excess of T cell disease. Twenty five (24%) had a "characteristic" hyperdiploid karyotype and as a group had lower presenting white counts, a tendency to CD10, and periodic acid schiff positivity of the blast cells and smaller spleens. None was an infant and only one was over 10 years old. Seven (7%) children with t(9; 22), t(8; 14), or t(4; 11) translocations were grouped together as "specific" translocations. Collectively they had a significantly worse prognosis than the remainder. Nine children developed central nervous system relapse, six of whom had either t(4; 11) or abnormalities of 9p or 19p. A descriptive classification taking into account chromosome bonding pattern is cytogenetically more appropriate and may be more clinically useful than grouping children simply by chromosome number. As knowledge and techniques improve, the classification of cytogenetic abnormalities in ALL will need to be kept under frequent review.

Nonendemic Burkitt's lymphoma with complex chromosome abnormalities involving chromosomes 2 and 8.

A long surviving patient with nonendemic Burkitt's lymphoma and complex cytogenetic findings is presented. Chromosome abnormalities were seen as a minor clone in peripheral blood and were considered consistent with the t(2;8)(p12;q24) variant. The karyotype was 47,XY, -2, -8, + der2(8qter-8q24::2p12-2qter), + der8(8pter-8q23::2p12-2pter) + der8(8qter-8cen::1q21-1qter). This case illustrates the value of extensive chromosome analysis in hematologic disorders and, at the time of writing, is the first example in Britain of the t(2;8) variant in Burkitt's lymphoma.

Methotrexate and bone marrow metaphases.

The efficacy of a methotrexate (MTX) block/thymidine release synchronization technique has been assessed in bone marrow cultures from patients with acute nonlymphocytic leukemia and myelodysplasia. In contrast to cultures of stimulated lymphocytes from normal individuals, no improvement in mitotic index (MI) or metaphase quality could be detected using this technique. Demonstration of an unchanged level of division in bone marrow cultures in the presence of MTX suggests that the technique is unsuitable for synchronization purposes in this tissue. The influence of preincubation prior to MTX exposure and duration of exposure to colcemid on MI and metaphase quality have also been examined.

Value of cytogenetic studies in prediction of acute phase CML.

Twenty-five patients with Ph CML who eventually developed a blast crisis were karyotyped at regular intervals in order to correlate the evolution of abnormal clones with clinical changes. Persistent new clones appeared in the chronic phase, prior to either transition or transformation, or in the acute phase (the latter, particularly, where transformation was slow). In many patients, chromosome changes accumulated within a single abnormal clone as the disease progressed. In others, particularly where the simple Ph cell line was slow to be supplanted, new clones appeared from the remaining Ph cells at the time of transformation, perhaps reflecting the inability of certain chromosome abnormalities to coexist. We suggest that the occurrence of additional chromosome abnormalities is not reliable evidence of acute transformation, but that the nature and subsequent behavior of abnormal clones may provide more valuable indications.

Trisomy 15 associated with loss of the Y chromosome in bone marrow: a possible new aging effect.

Trisomy 15 as a single autosomal abnormality is a rare finding in hematological disorders and has not as yet been associated with any specific disease type. We report 20 cases of trisomy 15 observed in the bone marrow of patients referred for a suspected hematological malignancy. Most patients were elderly, and a marked male predominance was evident. Aneuploidy for the Y chromosome was observed in addition to +15 in 11 out of 15 male patients. A myelodysplastic disorder (MDS) was confirmed in six cases, and acute myeloid leukemia (AML) in one. There was no evidence of malignant hematological diseases in the remaining 13 patients. We propose that there may be an association between loss of the Y chromosome and trisomy 15 and that trisomy 15, like missing Y, may not always be a marker of malignancy, but may reflect an underlying age effect. The possibility that its presence may herald the development of a malignant condition cannot, however, be excluded.

Chromosome studies in eleven colorectal tumors.

Cytogenetic analysis is presented on seven freshly derived colorectal tumors and four established cell lines (SW 742, SW 480, SW 948, and HT 29). No chromosome change was common to all tumors, although previous nonrandom findings were confirmed. Single chromosome abnormalities were identified in two cases, 47,XX,+i(7p) and 46,XX,-17,+der(17),t(17;?)(p;?), and their relevance to tumor origin and development is discussed. The association of i(8q) with tumors of the rectosigmoidal region is confirmed, and it is suggested that other rearrangements involving loss of 8p may have the same association. Abnormalities resulting in loss of 20p and duplication of 20q, not previously reported as a nonrandom change, were seen in seven out of 11 cases.

Cytogenetic analysis of a United Kingdom series of non-Hodgkins lymphomas.

We describe cytogenetic analyses of cells derived from 40 non-Hodgkins lymphoma (NHL) node biopsies, 23 of which were from patients who had not been treated before biopsy. We noted that the chromosomes most frequently gained were X (32%), 12 (27%), and 3 (24%). Monosomies were much less common; loss of chromosome 13 (13.5%) was most frequent. Structural abnormalities primarily involved chromosomes 14 (70%), 1 (40.5%), 18 (38%), 6 (35%), and 17 (22%). Low-and high-grade disease showed similar patterns of structural changes; however, a markedly greater number of chromosome gains were associated with low-grade disease. Biopsy samples from patients who had previously been treated showed an increased frequency of structural abnormalities, as well as a significantly larger number of chromosome gains. The importance of these observations, particularly with regard to possible oncogene involvement in lymphoma evolution, is discussed.

Blood-CSF barrier function in the rat embryo.

Blood-cerebrospinal fluid (CSF) barrier function and expansion of the ventricular system were investigated in embryonic rats (E12-18). Permeability markers (sucrose and inulin) were injected intraperitoneally and concentrations measured in plasma and CSF at two sites (lateral and 4th ventricles) after 1 h. Total protein concentrations were also measured. CSF/plasma concentration ratios for endogenous protein were stable at approximately 20% at E14-18 and subsequently declined. In contrast, ratios for sucrose (100%) and inulin (40%) were highest at the earliest ages studied (E13-14) and then decreased substantially. Between E13 and E16 the volume of the lateral ventricles increased over three-fold. Decreasing CSF/plasma concentration ratios for small, passively diffusing molecules during embryonic development may not reflect changes in permeability. Instead, increasing volume of distribution appears to be important in this decline. The intracellular presence of a small marker (3000 Da biotin-dextranamine) in plexus epithelial cells following intraperitoneal injection indicates a transcellular route of transfer. Ultrastructural evidence confirmed that choroid plexus tight junctions are impermeable to small molecules at least as early as E15, indicating the blood-CSF barrier is morphologically and functionally mature early in embryonic development. Comparison of two albumins (human and bovine) showed that transfer of human albumin (surrogate for endogenous protein) was 4-5 times greater than bovine, indicating selective blood-to-CSF transfer. The number of plexus epithelial cells immunopositive for endogenous plasma protein increased in parallel with increases in total protein content of the expanding ventricular system. Results suggest that different transcellular mechanisms for protein and small molecule transfer are operating across the embryonic blood-CSF interface.

Long-term changes in blood-brain barrier permeability and white matter following prolonged systemic inflammation in early development in the rat.

Epidemiological evidence in human fetuses links inflammation during development with white matter damage. Breakdown of the blood-brain barrier has been proposed as a possible mechanism. This was investigated in the present study by inducing a prolonged inflammatory response in newborn rats, with intraperitoneal injections of lipopolysaccharide (LPS; 0.2 mg/kg) given at postnatal (P) day 0, P2, P4, P6 and P8. An acute phase response was present over the whole period of injections. Changes in blood-brain barrier permeability were determined for small (sucrose and inulin) and large (protein) molecules. During and immediately after the inflammatory response, plasma proteins were detected in the brain only within white matter tracts, indicating an increased permeability of the blood-brain barrier to protein during this period. The alteration in permeability to protein was transient. In contrast, the permeability of the blood-brain barrier to 14C-sucrose and 14C-inulin was significantly higher in adult animals that had received serial LPS injections during development. Adult animals receiving a single 1 mg/kg LPS injection at P0 showed no alteration in blood-brain barrier permeability to either small or larger molecules. A significant decrease in the volume of CNPase immunoreactive presumptive white matter tracts occurred in the external capsule and corpus callosum at P9. These results demonstrate that a prolonged systemic inflammatory response in the early postnatal period in rats causes size selective increases in blood-brain barrier permeability at different stages of brain development and results in changes in white matter volume.

Breakdown of the blood-brain barrier to proteins in white matter of the developing brain following systemic inflammation.

Compromised blood-brain barrier permeability resulting from systemic inflammation has been implicated as a possible cause of brain damage in fetuses and newborns and may underlie white matter damage later in life. Rats at postnatal day (P) 0, P8 and P20 and opossums (Monodelphis domestica) at P15, P20, P35, P50 and P60 and adults of both species were injected intraperitoneally with 0.2-10 mg/kg body weight of 055:B5 lipopolysaccharide. An acute-phase response occurred in all animals. A change in the permeability of the blood-brain barrier to plasma proteins during a restricted period of postnatal development in both species was determined immunocytochemically by the presence of proteins surrounding cerebral blood vessels and in brain parenchyma. Blood vessels in white matter, but not grey matter, became transiently permeable to proteins between 10 and 24 h after lipopolysaccharide injection in P0 and P8 rats and P35-P60 opossums. Brains of Monodelphis younger than P35, rats older than P20 and adults of both species were not affected. Permeability of the blood-cerebrospinal fluid (CSF) barrier to proteins was not affected by systemic inflammation for at least 48 h after intraperitoneal injection of lipopolysaccharide. These results show that there is a restricted period in brain development when the blood-brain barrier, but not the blood-CSF barrier, to proteins is susceptible to systemic inflammation; this does not appear to be attributable to barrier "immaturity" but to its stage of development and only occurs in white matter.

Transformation of human cells by SV40 virus.

Fibroblast cultures were prepared from skin biopsies from 29 patients and tested for their susceptibility to transformation by simian virus SV40. Cells with a normal chromosome complement showed a mean transformation frequency of 25/106 cells but for cells from a single patient with Fanconi's anaemia, the value was 152/106 cells. An increased susceptibility to transformation was observed for cells from 6 patients with Down's syndrome 3 patients with trisomy 18, a patient with trisomy 18 for 5% of cells and a patient with trisomy 13. No increased susceptibility to transformation was found for cells with a chromosome complement of XO, XXY, XX/XX + 8, XX + partial 15q or XX + 9p. The susceptiability to transformation was related to susceptibility to SV40 virus infection, as measured by the number of infected cells which contained SV40 virus induced T antigen. This latter test was technically easier to perform and could serve to detect persons of increased susceptiability to transformation, since this may indicate an increased risk of natural malignant disease.

Karyotypic abnormalities in transformed chronic granylocytic leukaemia.

Chromosomal abnormalities in three cases of chronic granylocytic leukaemia are presented. The relative importance of 'specific' chromosomal abnormalities, additional to the Ph1 in the karyotypic evolution of chronic granylocytic leukaemia is discussed. A new abnormal metacentric chromosome is described.

Comparison of transformation and T antigen induction in human cell lines.

Skin fibroblast cultures were established from eight individuals. These cell cultures, together with WI-38 cells, were examined for susceptibility to transformation by SV40 virus. Four transformation-susceptible cell lines (TS), established from patients with Down's syndrome, were found to be three to four times more susceptible to transformation than transformation-resistant cell lines (TR) from normal individuals. TR and TS cell lines were compared for their susceptibility to induction of SV40 T antigen. For dividing cells T antigen was detected in a higher percentage of TS cells than TR cells. For nondividing cells, the reverse was found; T antigen was detected in 10-fold more cells of the TR lines than in cells of the TS lines. Similar results were obtained after infection of cells with CELO virus. Titration of vaccinia virus and influenza virus A2/Scotland/49/57 indicated that TR and TS cells were equally sensitive to the former virus, but TR cells were three to five times more sensitive to influenza virus A2/Scotland/49/57 than were TS cells.

Multiple congenital defects associated with trisomy for long arm of No. 4.

The clinical and cytogenetic findings of a male infant with multiple congenital anomalies and trisomy for the distal third of the long arm of No. 4 are described. The abnormal chromosome was inherited from the mother who had a balanced translocation, t(4;9)(q31;q34). Trisomy for the long arm of No. 4 has previously been described in only 3 patients.

Non-random and random chromosomal abnormalities in transformed chronic granulocytic leukaemia.

Chromosome abnormalities, identified using a banding technique and additional to the Ph, are reported in 10 consecutive cases of transformed chronic granulocytic leukaemia. In most of the cases the abnormalities were non random. In 2 cases serial studies were performed and additional abnormalities found, antedating transformation by one week and two months respectively. In 2 others the Ph status had been established during the chronic phase of the disease. In the remaining cases the first chromosome analysis was performed at the time of transformation.

Overestimation of pediatric cardiac output by thermal indicator loss.

The accurate measurement of pediatric cardiac output by thermodilution requires that the quantity of cold indicator introduced into the central circulation be known. This study defines an important source of error in the correction factor for the amount of heat gained by small volumes of cold injectate during passage through pediatric catheter systems. This error may result in significant overestimation of cardiac output (as much as 59%) when blood at body temperature is withdrawn into the injection lumen of the pediatric catheter before the injection.