Experimental transmission of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) to SCID-hu Thy/Liv mice.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is a novel human lymphotropic herpesvirus linked to several human neoplasms. To date, no animal model for infection by this virus has been described. We have examined the susceptibility of C.B-17 scid/scid mice implanted with human fetal thymus and liver grafts (SCID-hu Thy/Liv mice) to KSHV infection. KSHV virions were inoculated directly into the implants, and viral DNA and mRNA production was assayed using real-time quantitative polymerase chain reaction. This revealed a biphasic infection, with an early phase of lytic replication accompanied and followed by sustained latency. Ultraviolet irradiation of the inoculum abolished all DNA- and mRNA-derived signals, and infection was inhibited by ganciclovir. Viral gene expression was most abundant in CD19(+) B lymphocytes, suggesting that this model faithfully mimics the natural tropism of this virus. Short-term coinfection with HIV-1 did not alter the course of KSHV replication, nor did KSHV alter levels of HIV-1 p24 during the acute phase of the infection. Although no disease was evident in infected animals, SCID-hu Thy/Liv mice should allow the detailed study of KSHV tropism, latency, and drug susceptibility.

Human herpesvirus 6 (HHV-6) causes severe thymocyte depletion in SCID-hu Thy/Liv mice.

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that may act as a cofactor in the progression of AIDS. Here, we describe the first small animal model of HHV-6 infection. HHV-6 subgroup A, strain GS, efficiently infected the human thymic tissue implanted in SCID-hu Thy/Liv mice, leading to the destruction of the graft. Viral DNA was detected in Thy/Liv implants by quantitative polymerase chain reaction (PCR) as early as 4 d after inoculation and peaked at day 14. The productive nature of the infection was confirmed by electron microscopy and immunohistochemical staining. Atypical thymocytes with prominent nuclear inclusions were detected by histopathology. HHV-6 replication was associated with severe, progressive thymocyte depletion involving all major cellular subsets. However, intrathymic T progenitor cells (ITTPs) appeared to be more severely depleted than the other subpopulations, and a preferred tropism of HHV-6 for ITTPs was demonstrated by quantitative PCR on purified thymocyte subsets. These findings suggest that thymocyte depletion by HHV-6 may be due to infection and destruction of these immature T cell precursors. Similar results were obtained with strain PL-1, a primary isolate belonging to subgroup B. The severity of the lesions observed in this animal model underscores the possibility that HHV-6 may indeed be immunosuppressive in humans.

Ion channels in plasmalemma of wheat protoplasts.

The patch-clamp technique was used to study passive movements of ions through the plasmalemma of wheat leaf protoplasts. This method overcomes the problems inherent in conventional electrophysiological study of plant cells. Changes in conductance were recorded in patches excised from the plasmalemma. Two types of patches were observed: (i) regions of low channel density, where discrete single-channel currents could be resolved and conductance ranged from 10 to 200 picosiemens and (ii) regions of high channel density, where single-channel currents could not be resolved and conductance was on the order of a few nanosiemens. The results indicate a striking similarity between animal and plant cell membranes in the basic phenomena of transport. Moreover, the approach used constitutes a new degree of refinement in the study of processes of regulation, pathology, and toxicity in plants.

Interferometer experiment with independent local oscillators.

We have operated an interferometer with independent local oscillators and without any communication link of wide bandwidth between the elements of the interferometer. This makes operation possible at very long base lines because, heretofore, construction of the communications link has been the factor limiting the separation of the elements. In our system, coherence at the two elements is maintained through the use of two highly stable, atomic oscillators. The intermediate-frequency output signals are recorded at each element on a high-speed digital tape recorder. Interference fringes are produced later by cross-correlating the two tape records in a digital computer.

Spectral line interferometry with independent time standards at stations separated by 845 kilometers.

An upper limit of 0.02 second of arc has been determined for a hydroxyl radical (OH) emission region associated with the radio source W3, with the use of a Michelson interferometer consisting of two radio telescopes 845 kilometers apart. Timing was provided at the stations by independent atomic frequency standards. The 1665-megahertz radiation was translated to video frequency and recorded digitally on magnetic tapes which were later processed by computer, yielding fringe phase and amplitude as a function of frequency over the received bandwidth.

Suppression of T-cell responsiveness by inducible cAMP early repressor (ICER).

Depending on the nature of the costimulation of T lymphocytes, expression of regulatory cytokines and chemokines is either susceptible or resistant to cyclic AMP (cAMP)-mediated inhibition. Our data show that cAMP-mediated inhibition of endogenously expressed cytokines, which is characteristic for T helper (Th) 1- and Th 2-like phenotypes, correlates with the induction of a potent transcriptional repressor, inducible cAMP early repressor (ICER), in both subsets of T cells activated under conditions of suboptimal interleukin-2 (IL-2) expression. Importantly, Th-specific expression of certain chemokines is also susceptible to cAMP-mediated transcriptional attenuation. To determine whether ICER per se, rather than forskolin-mediated elevation of intracellular cAMP, is responsible for the observed inhibitory effect, we generated transgenic mice expressing ICER under the control of a lymphocyte-specific lck promoter. On stimulation, transgenic thymocytes overexpressing ICER exhibited reduced levels of IL-2 and interferon (IFN)-gamma and failed to express the macrophage inflammatory protein (MIP)-1alpha and MIP-1beta genes. Splenic T cells from ICER-transgenic mice showed a defect in proliferation and lacked a mixed lymphocyte reaction response, implying that ICER-mediated inhibition of cytokine and chemokine expression might play an important role in T-cell inactivation.

The critical role of nucleus accumbens dopamine systems in the mediation of fixed interval schedule-controlled operant behavior.

Microinjections of the irreversible dopamine antagonist EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline), into nucleus accumbens but not into dorsal striatum, markedly decreased response rates on a fixed interval schedule of reinforcement. These preferential effects in nucleus accumbens could be prevented by selectively activating either D1 or D2 receptors. These data demonstrate a critical role for mesolimbic dopamine systems in the mediation of fixed interval schedule-controlled operant behavior.

Thebaine content of selections of Papaver bracteatum Lindl. at different ages.

Approximately 200 accessions of Papaver species were evaluated for identification as P. bracteatum Lindl. and for thebaine content. Fifteen authentic P. bracteatum accessions were selected on the basis of chromosome count. Statistical analyses of variation in alkaloid content were made for these accessions. Four of the 15 warranted further study based on the vigor of the plant and the total thebaine present in the tissue. Significant variability in thebaine content was found in wild strains of P. bracteatum, thus requiring genetic selection studies. The data showed that total yield potential of thebain should be considered in selecting P. bracteatum strains for commercial growth rather than concentrating on strains that produce the highest thebaine concentrations. A method for the quantitative estimation of thebaine from P. bracteatum is presented.

B-mode and Doppler ultrasonography in pony mares with experimentally induced ascending placentitis.

REASONS FOR PERFORMING STUDY: Early, accurate diagnosis of ascending placentitis in mares remains a key challenge for successful treatment of the disease. Doppler ultrasonography has shown promise as a tool to diagnose pregnancy abnormalities and is becoming more available to equine clinicians. However, to date, no studies have prospectively compared this technique to standard B-mode measurement of the combined thickness of the uterus and placenta (CTUP). OBJECTIVES: The objective of the current study was to compare Doppler and B-mode ultrasonography for the detection of experimentally-induced ascending placentitis in mares. METHODS: Eleven healthy pony mares in late gestation were used in this study. Placentitis was induced in 6 mares between Days 280 and 295, while 5 mares served as negative controls. All mares were intensively monitored until delivery. Fetal heart rate, CTUP, uterine artery blood flow (resistance index, pulsatility index, arterial diameter and total arterial blood flow) and physical examination findings were recorded at each examination. Mares with an increased CTUP above published values were treated in accordance with published recommendations. Foals and fetal membranes were examined at birth. Ultrasonographic parameters were compared between groups using ANOVA. Foal viability and histological presence of placentitis were compared using a Fisher's exact test. RESULTS: The CTUP was increased above normal in 5 of 6 inoculated mares within 3 days after inoculation (P = 0.05). The sixth inoculated mare was excluded from subsequent data analysis. Uterine artery blood flow, physical examination findings and fetal heart rate were not different between groups. Gradual increases in CTUP, arterial diameter and total arterial blood flow were detected with increasing gestational age in the control mares (P = 0.02, P = 0.00001 and P = 0.00001, respectively). CONCLUSION: The CTUP, but not uterine blood flow, was different between groups (P = 0.00001). Recorded CTUP values for control pony mares were similar to previously published values for light breed horses.

Forecasting first births.

This study is concerned with one aspect of the family cycle, namely, the transition from young married to young married with small children. The focus is on developing models to forecast entries into this latter stage for the purpose of marketing research. "Using ordinary least squares, forecasting models were estimated for (1) total number of first births, (2) number of white first births, and (3) number of nonwhite first births." Models are estimated for both the United States and California using data from official sources.

Thebaine variations between germplasm sources within one collection of Papaver bracteatum Lindl.

Plants of Papaver bracteatum Lindl. derived from different germplasm sources within one Iranian collection were grown for 18 and 37 weeks in the greenhouse and controlled environment chambers. Gas-chromatographic analyses showed significant differences between thebaine concentrations of root tissue for 18-week-old plants, but not for 37-week-old plants. The two age groups did not differ in thebaine yield per unit of dry weight of root tissue. However, due to more profuse root growth, the total yield of thebaine from roots of the older plants was about four times that of the younger plants. Shoot thebaine concentrations differed significantly for 18- and 37-week-old plants. The over-all mean thebaine concentration for shoots of older plants was nearly twice that of younger plants. The variation in thebaine concentrations in these plants suggested genetic differences between the germplasm sources. Thus, domestication of P. bracteatum will require selection of germplasm from within and between collections, for plants of the greatest economic potential.

Rapid semi-quantitative spot test for determination of thebaine and differentiation of Papaver bracteatum from P. orientale and P. pseudo-orientale.

The alkaloids, orientalidine, oripavine, thebaine, narceine, narceine, codeine, papaverine, isothebaine, alpenigenine and morphine react with a solution of sulfuric acid containing molybdate to give specific colors. Using the acid solution permitted the differentiation of Papaver bracteatum latex from P. orientale latex and P. pseudo-orientale latex.

The human anti-murine xenogeneic cytotoxic response. II. Activated murine antigen-presenting cells directly stimulate human T helper cells.

Generation of a human T cell anti-murine xenogeneic response has previously been shown to be dependent on presentation of murine Ag by human APC. We have undertaken a series of experiments to better delineate the cellular defects that prevent effective production of IL-2 by human T cells upon direct exposure to murine stimulator populations. It was found that although resting human T cells cannot respond effectively to resting murine APC, they can respond to activated murine stimulator populations. Such APC activation could be mediated by murine granulocyte-macrophage-CSF or LPS that were associated with increased expression of B7-2 on the xenogeneic stimulating cell populations. Blocking studies with Ab provided further evidence that costimulation through CD28 played a critical role in the stimulation of human T cells by activated murine-stimulator cells in the production of IL-2. These results demonstrate the usefulness of this xenogeneic system in understanding human T cell-APC interactions and defining minimally sufficient T cell activation requirements. They further delineate the cellular level of deficient activation in the xenogeneic stimulation of human T cells by murine cell populations, and identify the potential importance of CD28/CTLA4 and its ligands in xenogeneic responses. These observations and concepts have implications for clinical efforts in xenografting.

Traumatic bilateral renal artery thrombosis.

Although unilateral traumatic renal artery thrombosis may be more common than was once thought, bilateral traumatic renal artery thrombosis is rare, with only thirteen previously reported cases. Time is of the essence in diagnosing this condition, so that in the proper clinical setting one may be able to proceed quickly to arteriography. However, the prognosis at present still remains poor.

Potassium Channels in Motor Cells of Samanea saman: A Patch-Clamp Study.

Leaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K(+), Cl(-) and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K(+) currents that flow during leaflet movement. Recordings in the "whole-cell" mode reveal depolarization-activated K(+) currents in extensor and flexor cells that increase slowly (t((1/2)) = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K(+) currents is adequate to account quantitatively for K(+) loss, previously measured in vivo during cell shrinkage. The K(+) channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light- and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.

Efficacy of T cell depletion of surgically resected cadaveric marrows by monoclonal antibodies: considerations in HLA-mismatched marrow transplantation.

The potential therapeutic benefits of allogeneic bone marrow transplantation (BMT) remain generally unavailable to patients who have diseases amenable to treatment by allogeneic BMT, but who lack an identifiable HLA-matched marrow donor. If graft versus host disease and graft rejection can be controlled, then the possibility of expanding allogeneic BMT to minimally HLA-matched or fully mismatched combinations exists and a "universal" donor marrow bank might be established. Towards this end, we have evaluated surgical harvest of cadaveric marrow, T cell depletion of such marrow (for prevention of graft-versus-host disease), influence of surgical harvest on final T cell content, and final cell yield. Marrow harvest was coordinated with the donation of other tissues or organs of cadaveric origin. In a series of twenty-two surgical vertebral body harvests, the initial marrow yield per vertebral body was 4.5 x 10(9) with four to six vertebral bodies per harvest. T cell depletion was evaluated by a limiting dilution assay. Since a combination of multiple monoclonal antibodies with specificity for T cell surface molecules provided greater reproducibility in the depletion of T cells from marrow than the utilization of any single antibody, a pool of monoclonal antibodies and complement were used to treat the marrow. The final cell yield per vertebral body was 2.1 x 10(9) for an average total yield of 9.3 x 10(9) cells per harvest. T cell content for each marrow following T cell depletion was less than 0.001%. These marrow have been cryopreserved as a bank of characterized donor marrow for use in HLA minimally matched or unmatched marrow transplantation.

IL-7 increases both thymic-dependent and thymic-independent T-cell regeneration after bone marrow transplantation.

Thymic-dependent differentiation of bone marrow (BM)-derived progenitors and thymic-independent antigen-driven peripheral expansion of mature T cells represent the 2 primary pathways for T-cell regeneration. These pathways are interregulated such that peripheral T-cell expansion is increased in thymectomized versus thymus-bearing hosts after bone marrow transplantation (BMT). This study shows that this interregulation is due to competition between progeny of these 2 pathways because depletion of thymic progeny leads to increased peripheral expansion in thymus-bearing hosts. To test the hypothesis that competition for growth factors modulates the magnitude of antigen-driven peripheral expansion during immune reconstitution in vivo, a variety of T-cell active cytokines were administered after BMT. Of the cytokines (interleukins) tested (IL-3, IL-12, IL-6, IL-2, and IL-7), IL-2 modestly increased peripheral expansion in the face of increasing numbers of thymic emigrants, whereas IL-7 potently accomplished this. This report also demonstrates that the beneficial effect of IL-7 on immune reconstitution is related to both increases in thymopoiesis as well as a direct increase in the magnitude of antigen-driven peripheral expansion. Therefore, the administration of exogenous IL-7, and to a lesser extent IL-2, abrogates the down-regulation in antigen-driven peripheral expansion that occurs in thymus-bearing hosts after BMT. These results suggest that one mechanism by which T-cell-depleted hosts may support antigen-driven T-cell expansion in vivo is via an increased availability of T-cell-active cytokines to support clonal expansion.

Thymic-independent T cell regeneration occurs via antigen-driven expansion of peripheral T cells resulting in a repertoire that is limited in diversity and prone to skewing.

Thymic regenerative capacity in humans decreases with age, suggesting that thymic-independent pathways of T cell regeneration may predominate during adulthood. Using a murine bone marrow transplantation model, we present evidence that thymic-independent T cell regeneration occurs primarily via expansion of peripheral T cells and is Ag driven since significant expansion of CD4+ or CD8+ transgenic (Tg+)/TCR-bearing cells occurs only in the presence of Ag specific for the TCR. Such expansion resulted in skewing of the regenerated repertoire with 40 to 65% of the regenerated CD4+ or CD8+ T cells expressing the Tg+/TCR in thymectomized hosts after bone marrow transplantation. In experiments in which nontransgenic population are used as T cell inocula, we noted decreased CD4 expansion when Class II MHC was blocked by mAb treatment in vivo, an CD8 expansion failed to occur in Class I MHC-deficient hosts providing evidence that T cell regeneration in thymic-deficient hosts largely occurs via TCR-MHC-mediated selection of peripheral T cell populations. This process results in a T cell repertoire comprised exclusively of T cells recently activated by the antigenic milieu of the host, with negligible numbers of residual "naive" cells bearing TCRs for Ags absent at the time of expansion. These findings have important implications for approached to enhance T cell regeneration in humans and provide evidence that vaccine strategies could skew the T cell repertoire toward a specific antigenic target if administered to thymic-deficient hosts during immune reconstitution.