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.

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.

Fluorescence in situ hybridization analysis of 25 cases of idiopathic myelofibrosis and two cases of secondary myelofibrosis: monoallelic loss of RB1, D13S319 and D13S25 loci associated with cytogenetic deletion and translocation involving 13q14.

To identify a commonly deleted region of 13q14 in idiopathic myelofibrosis (IMF), we used fluorescence in situ hybridization analysis to test for deletion of the RB1 and BRCA2 genes, and the microsatellite loci D13S319 and D13S25, in a series of 25 patients. A further two patients with myelofibrosis secondary to polycythaemia vera and essential thrombocythaemia with reciprocal 13q translocations were studied in an attempt to further define the CDR. Twenty out of 21 patients with a cytogenetically normal chromosome 13 failed to show allelic loss with any of the four probes. In contrast, all four cases with cytogenetic deletion of 13q14 and both cases with 13q translocations involving 13q14 exhibited loss of RB1, D13S319 and D13S25. Loss of the BRCA2 locus was present in a single case only. Our results indicate that cryptic deletions of the 13q14 in myelofibrosis are rare. In addition, the genetic loss associated with cytogenetic 13q14 deletions or reciprocal translocations involving 13q14 is large and encompasses the gene-rich region around RB1, D13S319 and D13S25.

Acute-phase cytokines IL-1beta and TNF-alpha in brain development.

The nervous and the immune systems share several molecules that control their development and function. We studied the temporal and spatial distribution of the immunoreactivity of two acute-phase cytokines, TNF-alpha and IL-1beta, in the developing sheep neocortex and compared it with the well-described distribution of fetuin, a fetal glycoprotein also known to modulate the production of cytokines by lipopolysaccharide (LPS)-stimulated monocytes and macrophages. TNF-alpha was present first at embryonic day 30 (E30) (term is 150 days in sheep) as a faint band of immunoreactivity between the ventricular zone and the primordial plexiform layer (preplate). IL-1beta was detected at the first appearance of the cortical plate (E35-E40). Both cytokines were present on both sides of the cortical plate, which contained fetuin-positive cells, but was free from cytokine staining. By E60, TNF-alpha immunoreactivity was less prominent than that of IL-1beta and was confined to the marginal zone and outer developing white matter; IL-1beta was present in the marginal zone and in two bands of immunoreactive cells, one at the border of the cortical plate/developing layer VI (cells of neuronal morphology) and the other at the border of layer V and the developing white matter (identified as microglia). By E80, TNF-alpha staining had disappeared and IL-1beta-immunopositive microglia were no longer detectable. By E100-E140 only a few immunoreactive cells were identified in layers V-VI; these did not co-localize with fetuin-positive cells. The differences in distribution between fetuin and the two cytokines suggest that the opsonizing role of fetuin, proposed for monocyte production of cytokines, is probably not present in the developing brain. However, early in neocortical development TNF-alpha and IL-1beta were present in the subplate zone at a time of intense synaptogenesis.

Association of specific chromosome alterations with tumour phenotype in posterior uveal melanoma.

Posterior uveal melanomas have recurrent alterations of chromosomes 1, 3, 6 and 8. In particular, changes of chromosomes 3 and 8 occur in association, appear to characterize those tumours with a ciliary body component, and have been shown to be of prognostic significance. The relevance of other chromosome alterations is less certain. We have performed cytogenetic analysis on 42 previously untreated primary posterior uveal melanomas. Of interest was the observation that as tumour size increased the involvement of specific chromosome changes, and the amount of chromosome abnormalities likewise increased. Loss, or partial deletions, of the short arm of chromosome 1 were found to associate with larger ciliary body melanomas; typically, loss of the short arm resulted from unbalanced translocations, the partners of which varied. Trisomy of chromosome 21 occurred more often in ciliary body melanomas, whilst rearrangements of chromosomes 6 and 11 were primarily related to choroidal melanomas. Our results imply that alterations of chromosome 1 are important in the progression of some uveal melanomas, and that other chromosome abnormalities, besides those of chromosomes 3 and 8, are associated with ocular tumours of particular locations.

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.

Abnormalities of chromosomes 3 and 8 in posterior uveal melanoma correlate with prognosis.

Posterior uveal melanomas have nonrandom alterations affecting chromosomes 3, 6, and 8. Loss of chromosome 3 in uveal melanoma has been shown to act as a predictor of disease-free and overall survival. To confirm the significance of chromosome 3 loss and to extend the observations to include those of the associated alterations of chromosome 8, we have conducted a cytogenetic analysis on a series of 42 tumours from patients with primary uveal melanoma who were followed up for a median of 31 months (range = 8-96 months). Abnormalities of chromosomes 3 and 8 were the commonest changes and were confirmed in 10 tumours using fluorescence in situ hybridization. Monosomy of chromosome 3 was found in 21 (50%) of the tumours, and 23 (54%) tumours had additional copies of 8q. Alterations of chromosomes 3 and 8 were found occurring together in 19 (45%) of the tumours and were significantly associated with a ciliary body component (P < 0.0001). Prognostic indicators and changes of chromosomes 3 and 8 were analysed for correlation with patient survival. Of the chosen parameters, only ciliary body involvement (P = 0.003), monosomy of chromosome 3 (P = 0.0007), and additional copies of 8q (P = 0.003) correlated with reduced survival. Evaluation of the dosage effect of additional copies of chromosome arm 8q showed a significant association with reduced survival (P = 0.0001), which was also predictive of a decreased disease-free interval (P = 0.01). Thus, the cytogenetic analysis of uveal melanoma may provide a valuable predictor of prognosis.

Frequency and importance of change in blast cell karyotype in relapsing childhood lymphoblastic leukemia.

Blast cell chromosome abnormalities at presentation in childhood acute lymphoblastic leukemia (ALL) are common, and different patterns are known to be related to outcome. In contrast, the frequency and importance of further changes at the time of relapse remain unclear. Blast cell karyotype evolution was therefore studied in a group of children with recurrent disease. Of 134 consecutive children diagnosed between 1982 and 1992, 31 had a marrow relapse, and 24 had complete cytogenetic studies at both diagnosis and the time of recurrence. Fourteen (58%) of the 24 showed additional chromosomal abnormalities at relapse, 5 (21%) retained abnormalities identical to those seen at diagnosis, and 5 (21%) remained cytogenetically normal. The 14 with additional changes had shorter first remissions and showed shorter survival after relapse compared with the others. These findings indicate that emergency of cytogenetically recognizable subclones during the progression of childhood ALL could be a marker of more resistant disease.

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.

Non-random abnormalities of chromosomes 3, 6, and 8 associated with posterior uveal melanoma.

We present ten cases of posterior uveal melanoma which were karyotyped after short-term culture. One tumour had a normal chromosome complement. The remaining nine tumours were cytogenetically abnormal, with chromosomes 3, 6, 8, 11, and 13 most frequently involved. Abnormalities of chromosome 13 were seen in two cases, chromosome 11 in three cases, and chromosomes 3, 6, and 8 in five cases. Four tumours, all derived from the ciliary body, demonstrated monosomy 3 and i(8q), confirming the involvement of these aberrations with a subgroup of uveal melanomas arising from the ciliary body.

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.

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.

Cytogenetic findings in six posterior uveal melanomas: involvement of chromosomes 3, 6, and 8.

Six posterior uveal melanomas were karyotyped after short-term culture. One had a normal chromosome complement; the remaining five had limited chromosome changes. Involvement of chromosomes 1 and 6 was noted in two and four cases, respectively, and three ciliary body tumours demonstrated both monosomy 3 and i(8q).

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.

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.