Assessment of donor T-cell function in cellular blood components by the CD69 induction assay: effects of storage, gamma radiation, and photochemical treatment.

BACKGROUND: Functional donor T-lymphocytes in blood components may cause a variety of transfusion complications. A flow cytometric assay based on the measurement of induced CD69 expression may be an alternative to cell proliferation methods in determining the functional status of these cells in blood components. STUDY DESIGN AND METHODS: Seven units of whole blood, RBCs, and platelet concentrates (PCs) were stored under blood bank conditions. Half of 3 PCs each were gamma-radiated or treated with UVA+psoralen; the other half served as controls. Samples were analyzed for phorbolester-induced expression of CD69 as an indicator of cell responsiveness and for exclusion of propidium iodide as a measure of cell membrane integrity and viability. RESULTS: CD69 inducibility and propidium iodide exclusion decreased exponentially (half-life, 3. 3 and 8.1 days, respectively) during cold blood storage. Irradiation and UVA+psoralen treatment of PCs immediately reduced CD69 inducibility to 21 percent (controls, 82%; p = 0.004) and 12 percent (controls, 95%; p = 0.0008), respectively. The proportion of cells capable of propidium iodide exclusion was similar in treated samples and controls, but it declined faster in the treated samples during subsequent storage. CONCLUSION: Flow cytometric measurement of CD69 induction can be adapted to provide quantitative assessment of T-cell function in blood components. Results obtained by the CD69 assay are in general agreement with those previously reported by use of proliferation methods; the assay may be useful for special applications in transfusion medicine.

'Modeling' relationships among HIV-1 replication, immune activation and CD4+ T-cell losses using adjusted correlative analyses.

OBJECTIVE: To model the relationships among HIV-1 replication, immune activation and CD4+ T-cell losses in HIV-1 infection. METHODS: Cross-sectional analysis of baseline data from the Viral Activation by Transfusion Study. Comparisons of unadjusted and adjusted correlative analyses to establish models for mechanisms of cell loss in AIDS. RESULTS: Using these analyses, significant correlations were found among plasma levels of tumor necrosis factor alpha (TNFalpha) and its type two receptor (TNFrII), interleukin-6 (IL-6), beta2-microglobulin, expression of CD38 and HLA-DR on CD8+ T lymphocytes and plasma levels of HIV-1 RNA. When correlations among these indices were adjusted for possible intermediary correlations, the relationship between HIV-1 RNA levels and all plasma markers of immune activation could be accounted for by the correlation between plasma HIV-1 RNA and plasma TNFrII levels. In addition, the negative correlations that both HIV-1 RNA levels and TNFrII levels had with CD4+ T-cell counts were partially accounted for by the correlations of HIV-1 RNA and TNFrII with CD38 expression on CD8+ T cells. In persons with advanced disease (CD4+ T cells < 50 x 10(6)/l) IL-6 levels were inversely correlated with CD4+ T-cell counts. CONCLUSIONS: This analysis is consistent with a model wherein HIV-1 replication induces TNFalpha expression that induces multiple other indices of immune activation. In this model, HIV-1 replication and TNFalpha expression induce CD4+ T-cell losses at least in part through mechanisms reflected in heightened CD38 expression.

Immune response to blood transfusion invery-low-birthweight infants.

BACKGROUND: Allogeneic blood transfusion is common in the treatment of neonatal anemia of prematurity or anemia due to multiple phlebotomies. The immune response of neonates to passenger leukocytes from allogeneic red cells was investigated. STUDY DESIGN AND METHODS: Fourteen infants (4 male, 10 female) prospectively were randomly assigned to receive either white cell-reduced (Group 1) or non-white-cell reduced (Group 2) irradiated blood. Blood samples were taken before and at various time intervals after transfusion (Days 1, 5-7,and 10-14). Cord blood from 11 healthy term infants was used for comparison. The following surface markers were used to assess immune modulation by flow cytometry: CD45RA/CD45RO, CD4/CD8, CD25/CD28, CD3/DR, CD14/B7, and CD3/CD56+CD16. Donor cell microchimerism was studied using semi-quantitative polymerase chain reaction Y-chromosome detection in female infants who received male donor blood. Donor and recipient HLA class II typing was performed with polymerase chain reaction with sequence specific primers. RESULTS: The lymphocyte counts in both groups were significantly increased after transfusion, and there was a significant increase in lymphocytes expressing CD45RA, CD3-/CD16+CD56, CD80, and CD3-/DR on Day 14. The premature infants' pretransfusion natural killer cell population (CD3-/CD16+CD56) was significantly lower than that of term infants, but it reached a similar level by Days 10-14. CD8 subpopulations were increased but not CD4+ cells. Two female infants (of 6) had circulating Y chromosomes 1 day after transfusion, and most of the infants effectively cleared the donor cells within 24 hours of transfusion. Two Group 2 infants who by chance received presumably HLA-haploidentical donor blood developed necrotizing enterocolitis. CONCLUSION: Blood transfusion alters immune cell antigen expression in premature neonates and may initially be immunostimulatory and later immunosuppressive.

Quantitation of white cell subpopulations by polymerase chain reaction using frozen whole-blood samples. Viral Activation Transfusion Study.

BACKGROUND: Previous methods for processing whole blood (WB) for nucleic acid analyses of white cells (WBCs) required fresh blood samples. A simple protocol that involves the freezing of WB for quantitative polymerase chain reaction (PCR) analyses was evaluated. STUDY DESIGN AND METHODS: Controlled studies were conducted in which paired fresh and frozen WB preparations were analyzed. The integrity of WBCs in the frozen WB samples was first assessed by flow cytometry using CD45 fluorescence, and calibration beads to quantitate recovery of WBC subsets. PCR of an HLA-DQ-A sequence was used to quantitate residual WBCs in a double-filtered red cell (RBC) component spiked with serial dilutions of WBCs, as well as in 51 filtered RBCs and 19 filtered platelet concentrates. Y-chromosome-specific PCR was used to quantitate male WBCs in five female WB samples spiked with serial dilutions of male WBCs and in serially collected frozen WB samples from four females transfused with male blood components. RESULTS: By flow cytometry, all major WBC subpopulations in frozen-thawed WB were quantitatively recovered and immunologically intact, although they were nonviable. HLA-DQ-A PCR quantitation of a dilution series from 8 to 16,700 per mL of WBCs spiked into double-filtered RBCs showed linear correlation of the results with both fresh and frozen preparations of the expected WBC concentrations (r2 = 0.98, p<0.0001 for both), without significant difference between observed and expected values (p>0.05). Y-chromosome-specific PCR results in female WB samples spiked with male WBCs were not significantly different in fresh and frozen preparations over a 3 log10 range of male cells. The results of WBC survival studies on frozen WB samples were consistent with previous observations in fresh blood samples. CONCLUSION: Direct freezing of WB enables subsequent recovery of WBCs for quantitative PCR analyses, with results comparable to those of fresh preparations.This protocol should facilitate wider implementation of nucleic acid-based analyses for quality control of WBC-reduced components, as well as for prospective clinical studies of microchimerism in transfusion and transplant recipients.

Safety of the blood supply.

Three of four recipients of transfusion in the United States are patients undergoing surgery, and despite promising advances in the development of alternatives to allogeneic blood transfusion, it is likely that for years to come this patient population will remain dependent on blood donated by volunteers. The safety of the blood supply has been questioned seriously since it became known that the human immunodeficiency virus could be transmitted by transfusion. In response to this threat, enforcement of strict donor eligibility criteria, removal of high risk donors from the donor pool, and testing of each donation with a panel of viral markers were instituted which have reduced the infectious risks of allogeneic blood transfusion dramatically during the last decade. The current safety of the blood supply is reviewed and the ongoing efforts to improve the safety of transfusions in the future are summarized briefly.

Lymphocyte subset analysis on frozen whole blood.

An approach to perform lymphocyte subset analysis on frozen-thawed whole blood (F/T WB) is described. WB from 24 human immunodeficiency virus type 1 (HIV-1) seropositive individuals and 21 controls was analyzed fresh and after frozen storage (with or without dimethyl sulfoxide) at -80 degrees C, in liquid nitrogen (LN2), and at -20 degrees C. Analysis of F/T WB utilized 3-color flow cytometry with CD45 and right angle light scatter gating. Absolute cell counts were obtained for 30 samples by using staining tubes containing internal bead standards [TruCount, Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA]. The mean difference between CD3+4+ percentages for F/T (-80 degrees C storage for up to 1 year) and fresh WB was less than -0.2% (95% limits +/-3%, P = 0.5) with 39 of 45 (87%) results falling within 2% of the fresh values (P = 0.74). Absolute CD3+4+ cell counts for F/T WB were generally lower than corresponding results for fresh aliquots (median difference was 33 cells/microl, P < 0.0001), but the results were highly correlated (r2 = 0.975, P < 0.0001). Results were more variable, although still highly correlated, for CD3+8+ cells, and with other freezing and storage conditions. It is concluded that lymphocyte subset analysis using F/T WB yields comparable results to fresh samples, which should prove useful for a number of practical applications.

Effect of storage and ultraviolet B irradiation on CD14-bearing antigen-presenting cells (monocytes) in platelet concentrates.

BACKGROUND: Ultraviolet B (UVB) irradiation of platelet concentrate (PCs) reduces platelet alloimmunization, but the mechanism of the effect is unclear. Evidence suggests that UVB may downregulate the expression of surface adhesion molecules on passenger antigen-presenting cells in PCs. STUDY DESIGN AND METHODS: The effect of blood bank storage, platelet preparation from whole blood, and UVB irradiation on the quantitative expression of intercellular adhesion molecule-1 (ICAM-1, or CD54), HLA-DR, CD45, and CD11c on CD14-positive antigen-presenting cells (monocytes) was studied by using two-color flow cytometry. RESULTS: Blood bank storage for 4 days resulted in upregulation of ICAM-1 and HLA-DR and downregulation of CD14 but left the expression of CD11c and CD45 unchanged. Preparation of PCs from fresh whole blood was associated with a rapid increase in CD11c without upregulation of ICAM-1 and HLA-DR. UVB irradiation before storage inhibited the upregulation of ICAM-1 and HLA-DR, resulted in accelerated downregulation of CD14, and was associated with increased loss of monocytes. Agitation of the PC bag during irradiation was of critical importance, since omission of agitation resulted in largely uninhibited upregulation of ICAM-1 but was still associated with significantly higher cell loss than that seen in unirradiated controls. CONCLUSION: UVB exposure nonspecifically affects monocytes in PCs, resulting in downregulation of surface molecules that are important for antigen presentation, as well as in significant cell loss.

Rapid leukocyte accumulation by "spontaneous" rolling and adhesion in the exteriorized rabbit mesentery.

Leukocyte concentration in venules of exteriorized rabbit mesentery was calculated from intravascular white blood cell counts and venular length and diameter measured in histological whole mount preparations. Immediately after exteriorization, the mean venular leukocyte concentration (MVLC) was found to be slightly below systemic leukocyte concentration. One to five minutes later, MVLC was 7 times, and 30 minutes later, 20 times above systemic values. The concentrations of both polymorphonuclear and mononuclear leukocytes (PMNs, MNs) increased, but PMNs accumulated faster and in higher numbers (8 fold vs. 4 fold at 5 minutes, 24 vs. 14 fold at 30 minutes, respectively). The observed leukocyte accumulation in mesentery venules was found to be due to adherence of leukocytes which is absent in normal tissue, but was rapidly induced by exteriorization. Intravital microscopy revealed that many leukocytes were rolling along the venular endothelium. Dextran sulfate, a potent inhibitor of leukocyte rolling largely prevented leukocyte accumulation after exteriorization (twice systemic vs. 6 times in control). A monoclonal antibody (60.3) blocking CD18 of the leukocyte adhesion complex attenuated leukocyte accumulation to a lesser extent (3.5 times systemic). The data indicate that leukocyte rolling accounts for the major part of "spontaneous" leukocyte accumulation in the exteriorized mesentery.

Cell groups afferent to the telencephalon in a cartilaginous fish (Platyrhinoidis triseriata). A WGA-HRP study.

Cell groups projecting to the telencephalon in the guitarfish Platyrhinoidis triseriata were localized by retrograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) from injections into the basal forebrain bundle. Labeled neurons were observed bilaterally in the dorsal and ventral thalamus, hypothalamus, mesencephalon and rostral rhombencephalon. A comparison of the labeled cell groups to those which project to the telencephalon in terrestrial vertebrates reveals many parallels, including position in the brain, similarities in the afferent connections and immunoreactivity. Further data are needed to decide whether the cell groups in Platyrhinoidis are homologous or homoplasious to their counterparts in land vertebrates.

Connections of the corpus cerebelli in the thornback guitarfish, Platyrhinoidis triseriata (Elasmobranchii): a study with WGA-HRP and extracellular granule cell recording.

The neuronal connections of the cerebellar corpus in the guitarfish Platyrhinoidis triseriata were investigated by WGA-HRP injections and extracellular recording of sensory evoked electrical activity. Injections of WGA-HRP into the corpus resulted in retrograde labeling of the following cell groups bilaterally: pretectal and accessory optic nuclei, interstitial nucleus of Cajal, nucleus ruber, oculomotor and possibly trochlear nucleus, central (periaqueductal) gray, nucleus H, reticular formation of the midbrain, cerebellar nucleus, caudal part of nucleus F, tentatively locus coeruleus and subcoeruleus field, octaval and trigeminal nuclei, intermediate octavolateralis nucleus, medial inferior reticular formation, lateral reticular nucleus, and spinal cord. Unilaterally labeled cells were seen in the contralateral inferior olive, which was found to project in sagittal zones onto the molecular layer of the corpus. Terminal fields of efferent Purkinje cell axons were labeled over the ipsilateral cerebellar nucleus exclusively. Purkinje cells in different parts of the corpus project topographically onto subdivisions of the nucleus. Mapping of evoked electrical multiple unit activity recorded from the granule cell layer of the corpus shows separate visual and tactile areas, mostly confined to the anterior and posterior lobes, respectively. Granule cells within the tactile area also responded to lateral line stimuli and, at two distinct medial locations in the caudal and rostral parts of the posterior lobe, to weak electric field stimulation in the bath. The body surface is somatotopically represented in the tactile area, but discontinuities in the map might indicate that the somatotopy is "fractured".

Eye movements evoked from telencephalic stimulations in the piranha (Serrasalmus nattereri).

The telencephalon of spinally transsected, unanesthetized piranhas was systematically stimulated with microelectrodes to scan for eye movement inducing (EMI) sites which were then identified by histological means. EMI sites were found clustered in several distinct central and medial telencephalic areas. Various types of eye movements were evoked from these sites often in combination with movements of the jaw, gills and pectoral fins. It is concluded that eye movements evoked from the telencephalon may be mediated by the optic tectum.

Central connections of the olfactory bulb in the goldfish, Carassius auratus.

The central connections of the goldfish olfactory bulb were studied with the use of horseradish peroxidase methods. The olfactory bulb projects bilaterally to ventral and dorsolateral areas of the telencephalon; further targets include the nucleus praeopticus periventricularis and a caudal olfactory nucleus near the nucleus posterior tuberis in the diencephalon, bilaterally. The contralateral bulb and the anterior commissure also receive an input from the olfactory bulb. Contralateral projections cross in rostral and caudal portions of the anterior commissure and in the habenular commissure. Retrogradely labeled neurons are found in the contralateral bulb and in three nuclei in the telencephalon bilaterally; the neurons projecting to the olfactory bulb are far more numerous on the ipsilateral side than in the contralateral hemisphere. Afferents to the olfactory bulb are found to run almost entirely through the lateral part of the medial olfactory tract, while the bulb efferents are mediated by the medial part of the medial olfactory tract and the lateral olfactory tract. Selective tracing of olfactory sub-tracts reveals different pathways and targets of the three major tract components. Reciprocal connections between olfactory bulb and posterior terminal field suggest a laminated structure in the dorsolateral telencephalon.

An isthmo-optic system in a bony fish.

This investigation reveals the existence of an isthmo-optic system in a bony fish for the first time. Two cell aggregates of the isthmic region project bilaterally to each eye in Polypterus. The crossed connections are significantly more developed than the uncrossed ones. These findings provide further evidence for the presence of bilaterally projecting isthmo-optic systems in early stages of vertebrate evolution. Furthermore, they suggest that a loss of one or all of these connections during evolutionary or ontogenetic development reflects a parcellation process as proposed by the parcellation theory.

Afferent connections of the optic tectum in the piranha (Serrasalmus nattereri).

Injections of horseradish peroxidase into the optic tectum of the piranha resulted in retrograde transport to the following structures bilaterally: the central telencephalic nucleus, four hypothalamic nuclei, the caudal part of the dorsomedial optic nucleus, the ventral nucleus of the torus semicircularis, the torus longitudinalis, the perilemniscal nuclei, the reticular formation, and a tentatively identified locus coeruleus. In addition, labeled cells were found unilaterally in the contralateral tectum and in the medial octavolateralis nucleus, the ipsilateral thalamic portion of the dorsomedial optic nucleus, the corpus geniculatum laterale ipsum, the pretectal nucleus, the nucleus corticalis, the dorsal nucleus of the torus semicircularis, and the nucleus isthmi. Efferent projections of the optic tectum appeared identical to those reported in Holocentrus (Ebbesson and Vanegas 1976).

Voluntary ocular tracking movements in response to a sinusoidally moving intermittently active sound source.

Voluntary eye movements in response to a sinusoidally moving, intermittently active sound source were investigated in 40 young healthy human subjects. Visual afferent input was prevented during the experiments. With increasing burst repetition rates of the sound source, different forms of eye movements were observed: a) so-called single and repetitive adversive movements consisting of saccades which occur with certain latencies in response to single bursts; b) so-called saccadic eye tracking movements which are characterized by numerous small saccades occurring in accordance with the target (sound source) movement but lacking a time correlation between the saccades and single bursts. It was found that saccadic eye tracking movements were performed instead of adversive movements when at least 4 to 10 bursts per movement period of the sound source was transmitted (with movement frequencies of the sound source from 0.1 to 0.5 Hz). Slight intersaccadic drifts, which could be regularly observed in all types of eye movements were interpreted as instability of fixation in darkness.

A note on the reciprocal connections between the retina and the brain in the puffer fish Tetraodon fluviatilis.

This investigation employed the horseradish peroxidase methodology to trace afferent and efferent connections of the retina in the puffer fish Tetraodon fluviatilis. The retinal projections to the CNS are within the range described in other teleost fishes. In addition to this observation, 4 structures were identified in the CNS which project to the retina. These are the optic tectum, the dorsomedial optic nucleus, the pretectal nucleus and the large and well differentiated corpus geniculatum laterale ipsum of Meader.

Acoustic-induced eye movements.

This article reports the influence of moving acoustic signals on eye movements (oculomotor functions) using 350 healthy test subjects. An acoustic test setting developed especially for this purpose has made possible the application of moving acoustic signals of varying frequency and form (square stimuli, sinusoidal stimuli, circular movements), as well as the examination of visual-acoustic and vestibular-acoustic interactions. It must be stressed that according to these investigations, both voluntary and involuntary eye movements can be demonstrated in response to moving acoustic signals. Involuntary eye movements, however, under th given experimental conditions, only are observed in about 20% of all test subjects and their frequency of occurrence is highest in young female subjects. They are most pronounced in darkness and when there is no fixation, and they can be coupled to varying degrees with the given acoustic signal. Voluntary and involuntary eye movements do not differ very much from one another. They consist mainly of a series of saccades that form staircase jerks. In both cases, no typical smooth movements or nystagmoid movement forms occur--which are predominant in tracking eye movements of visual stimuli. Occasionally, slight drifting movements can be demonstrated in the intersaccadic intervals. At a larger movement amplitude and increased movement frequency of the sound signal, pure opposite jerks finally appear, which follow up to a movement frequency of 1 Hz. The eye movements are influenced only slightly by a change in the amplitude of the sound movement (+/- 15 degrees to +/- 90 degrees). However, a close phase relationship between eye and sound movement exists. If instead of continuous acoustic signals (music), single burst signals are given at an increasing repetition rate, this results (at low repetition rates of bursts) in orienting reactions upon the individual signals, while at high signal repetition rates (above 0.5 Hz), continuous eye movements in the form of staircase jerks result. In investigating acoustically induced tracking eye movements, many new questions have arisen that previously have been open only to psychophysical studies.

Bilaterally asymmetrical uptake of [14C]2-deoxyglucose by the octavo-lateralis complexes in flatfish.

[14C]2-deoxyglucose uptake by neurons located in the octavolateralis complex of adult flatfish is asymmetrical on the two sides of the brain. It appears that the neuronal activity on the side oriented upward is higher than that on the side of the brain facing downward. This finding may be significant with respect to the mechanisms of metamorphosis of flatfish and may account for the peculiar fact that these animals swim on one body side during adult live.

Form and accuracy of voluntary ocular tracking movements in response to sinusoidally moving acoustic targets.

Form and accuracy of voluntary ocular tracking in response to a horizontal sinusoidally moving auditory stimulus were examined in complete darkness. Forty young, healthy subjects participated in the study. Ocular movements under the applied experimental conditions consisted of a succession of saccades whose direction corresponded to the motion of sound. Though slight intersaccadic drifts were observed, pure smooth pursuit movements could not be recorded. A comparison of the amplitude of sound motion and the corresponding ocular motion showed the latter to be rather inaccurate. Independent of the actual amplitude of the sound motion (+/- 15 degrees up to +/- 90 degrees) the amplitude of the ocular movements, in most cases, remained constant within a range of approximately +/- 35 degrees to +/- 45 degrees. The accuracy in respect to phase relationship between target movement and ocular response was quite good. In response to sinusoidal motion of the sound, the ocular movements usually occurred simultaneously up to 0.5 Hz.