Adenovirus type 7 induces interleukin-8 in a lung slice model and requires activation of Erk.

Adenovirus (Ad), particularly Ad type 7 (Ad7), causes severe lung infection and pneumonia. Initially, Ad causes neutrophilic inflammation of the distal airways and alveoli. Interleukin-8 (IL-8) is the major lung neutrophil chemotaxin, and we have shown that Ad7 induces IL-8 release from the A549 alveolar epithelial cell line. We sought to determine whether ex vivo human and bovine lung tissue containing primary pneumocytes could be used as a more accurate and relevant model to study Ad acute inflammation. We found that cultured lung tissue preserved normal lung architecture for more than 10 days. IL-8 was generated upon exposure of the lung organ culture to Ad7. IL-8 production required activation of the Ras/Erk pathway, since a pharmacological inhibitor blocked the appearance of IL-8 in the medium. Both human and bovine lung explants supported replication of Ad7, and immunohistochemistry experiments demonstrated the presence of the Ad hexon antigen within alveolar epithelial cells. These findings show that our novel human lung organ culture accurately reproduces the in vivo infectious disease process. Thus, this organ culture model represents a valuable tool for studying the acute innate immune response to respiratory infections.

Tapeworm identification in the fat sand rat (Psammomys obesus obesus).

The identification of a tapeworm (Rodentolepis nana, formerly named Hymenolepis nana) infection in a research breeding colony of sand rats (Psammomys obesus obesus) was complicated because of the unexpected long length (< 150 mm) of the worms. Other morphologic features that were consistent with this identification included the number (24), size (16 mm), and shape of the hooks on the rostellum. No evidence of intermediate hosts was found in the colony. Previous surveys of natural populations of sand rats had not identified this tapeworm. However, a detailed search of the literature revealed that variation in the size of R. nana had been reported, thus supporting the final identification of the tapeworm. R. nana is important and interesting because of its zoonotic potential and because it is the only tapeworm that is able to infect its definitive host without use of an intermediate host. This report is presented to help clarify the ambiguity found in the laboratory animal literature about the differences in the size of R. nana among rodent species used in research.

Effects of citrated whole blood transfusion in response to hemorrhage.

BACKGROUND AND PURPOSE: Standard treatment for massive hemorrhage in dogs is infusion of whole blood or of packed red blood cells with fresh frozen plasma if whole blood is not available. Although most whole blood is collected using a citrate-based anticoagulant, knowledge of citrate's relevant non-anticoagulant effects is not widespread. Citrate's anticoagulant activity is achieved through chelation of divalent metal cations (e.g., magnesium, calcium), which may exacerbate cardiovascular and metabolic insults attributable to hemorrhage. METHODS: Blood pressures, gas tensions, metabolites, and electrolytes; myocardial metabolites, pressures, and contractility; cardiac output; and left cranial descending and circumflex coronary artery flows were measured in 21 anesthetized dogs after hemorrhage was induced by collection of blood into a citrated reservoir to mean arterial pressure of 45 mm Hg for approximately 60 min (until arterial lactate concentration was 7.0 mmol/L), followed by a 1-h transfusion and 2 h of maintenance. RESULTS: Arterial ionized calcium concentration, total peripheral resistance, and myocardial function decreased significantly during hemorrhage. All aforementioned responses but myocardial function continued to decrease during the initial 20 min of transfusion, then began to recover. Total peripheral resistance and end-systolic elastance were the only factors significantly related to calcium concentration. CONCLUSION: Transfusion with citrated whole blood may significantly alter calcium concentration, negatively affecting myocardial and vascular function.

The mechanism of activation of NK-cell IFN-gamma production by ligation of CD28.

We have investigated the mechanism by which anti-CD28 antibodies activates IFN-gamma production by murine NK cells. These studies reveal that engagement of CD28 alone by this antibody is a poor activator of this cytokine response. Effective stimulation requires simultaneous ligation of the receptor for Fc (FcgammaRIII, CD16) which on its own is also a poor inducer of murine NK cells. The mechanism by which immobilized anti-CD28 increases IFN-gamma mRNA abundance involves both upregulation of transcription as well as induction of mRNA stabilization. However, the elevation of transcription is not as evident as that induced by IL-12 which, in contrast, does not induce message stabilization. Thus ligation of CD28 in the presence of IL-12 results in a synergistic increase in production of the cytokine. Using this assay we have also determined that immobilized anti-CD28 cannot induce resting NK cells to produce IFN-gamma. In contrast, the same cells can be induced by BCL1-C11 tumor cells that express high amounts of the CD28 ligand, B7-2. These studies provide important insights into the ability of cells bearing counter-receptor for CD28 to activate NK cell-cytokine production in vivo.

Large-pore hemodialysis in acute endotoxin shock.

OBJECTIVE: This study was undertaken to test the hypothesis that hemodialysis with a large-pore membrane would improve heart function during acute endotoxin shock. SETTING: Large animal laboratory. DESIGN: Eighteen mongrel dogs were instrumented to measure left ventricular maximum end-systolic elastance (left ventricular maximum elastance at end systole), cardiac output, circumflex artery blood flow, and myocardial mechanical efficiency (CO x MAP/MVO2, where CO is cardiac output, MAP is mean arterial pressure, and MVO2 is myocardial oxygen consumption). Plasma catecholamine concentrations were determined by high-performance liquid chromatography. Endotoxin shock was induced by infusing 5.0 microg/kg/min of Escherichia col 0127:B8 endotoxin in the portal vein for 60 mins, followed by 2.0 microg/kg/min of constant infusion. Control dogs (n = 6) received 4.0 mL/kg/min of saline; hemodialysis dogs (n = 6) underwent venovenous hemodialysis in 50-min intervals using a polysulfone filter (1.2 m2; mean pore size, 0.50 nm; blood flow rate, 400 mL/min; ultrafiltrate, "zero-balanced"); shams (n = 5) were treated identically to hemodialysis dogs, except that no convective dialysis was performed. A fourth group (n = 6) was treated with dopamine (5.0-7.0 microg/kg/min, optimal dose for contractile increase based on dose-response studies). MEASUREMENTS AND MAIN RESULTS: After 2 hrs of treatment, left ventricular maximum elastance at end systole increased and was unchanged in controls (30 +/- 5 mm Hg/mm) and shams (24 +/- 6 mm Hg/mm) compared with basal control. Hemodialysis treatment increased contractility (53 +/- 4 mm Hg/mm), as did dopamine treatment (54 +/- 7 mm Hg/mm). Endotoxin shock reduced mechanical efficiency to 45% of basal control; with hemodialysis treatment, left ventricular efficiency returned to 64% of basal control measurement, compared with 49% with dopamine treatment. During treatment, myocardial glucose uptake was increased with hemodialysis compared with other groups. No difference was observed among groups for left ventricular end-diastolic pressures or dimensions, or catecholamine concentrations. CONCLUSIONS: Large-pore hemodialysis increased left ventricular contractility to a similar degree as dopamine and provided a marginal improvement in myocardial glucose uptake and mechanical efficiency.

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome.

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

Nitric oxide as a regulator of embryonic development.

The role of nitric oxide (NO) in activation of cGMP is well established. It has been proposed that the ratio of cAMP to cGMP may be important in the regulation of preimplantation embryonic growth and differentiation. Therefore, we determined the ability of murine preimplantation embryos to produce NO. In addition, NO as an endogenous smooth muscle relaxant and vasodilator is a candidate for involvement in embryo implantation because this process requires increased vascular permeability and uterine quiescence at the sites of blastocyst apposition. Nitrite assays, an indirect measure of NO production, indicate that preimplantation murine embryos produce NO. This production was reversibly inhibited by culture of embryos in medium containing a nonspecific NO synthase (NOS) inhibitor (NG-nitro-L-arginine). Additionally, inhibition of normal development was observed in embryos cultured with NOS inhibitor. NO levels increased in culture medium when ovariectomized progesterone-treated animals were exposed to estrogen for 1 h in utero. Such hormonal treatment induces implantation. These data indicate that NO levels are regulated by estrogen and may be important in regulation of implantation. In addition, these data demonstrate for the first time that NO production appears to be required for normal embryonic development.

Relationship between inspiratory pressure and tidal volume in the anesthetized canine.

Hounds undergoing prolonged or complicated surgical procedures are often underventilated, as indicated by blood gas and end-tidal CO2 (CO2) values when using published ventilatory guidelines. We investigated the relationship between body weight, tidal volume, and inspiratory pressure delivered by the ventilator (lung inflation pressure) in 59 anesthetized hounds (19 to 33 kg). Animals were ventilated under positive pressure control and noninvasively instrumented to monitor blood pressure, ECG, oxygen saturation, CO2, and tidal volume. Weight, sex, and thorax measurements were recorded. All dogs were monitored at lung inflation pressures of 10, 14, and 18 cm H2O, with measurements recorded once CO2 stabilized. Veterinary guidelines recommend tidal volumes of 10 to 15 ml/kg of body weight and lung inflation pressures of 15 to 25 cm H2O. When inflation pressure was below guidelines (10), tidal volume was "normal" (10 to 15 ml/kg), but the animals were underventilated. When inflation pressure was "normal" (14 or 18 cm H2O), tidal volume was above guidelines. Physiologic variables were normal only when inflation pressure was 14 cm H2O. Weight and thorax depth accounted for 32 and 6%, respectively, of tidal volume variability, and tidal volume varied by +/- 250 ml at any given body weight and inflation pressure. None of the measured physical variables accurately predicted tidal volume. These data suggest that the inconsistency in tidal volume is due to a previously undescribed variability in respiratory compliance in the anesthetized hound and that the guidelines for ventilation during surgery need further investigation.

Homodimerization of tumor-reactive monoclonal antibodies markedly increases their ability to induce growth arrest or apoptosis of tumor cells.

Monoclonal antibodies (mAbs) that exert antitumor activity can do so by virtue of their effector function and/or their ability to signal growth arrest or cell death. In this study, we demonstrate that mAbs which have little or no signaling activity-i.e., anti-CD19, CD20, CD21, CD22 and Her-2-can become potent antitumor agents when they are converted into IgG-IgG homodimers. The homodimers exert antigrowth activity by signaling G0/G1 arrest or apoptosis, depending upon which cell surface molecule they bind. This activity is specific and, in the case of the anti-CD19 mAb, did not require an Fc portion. These results offer the possibility that homodimers of other tumor-reactive mAbs which have little antitumor activity as monomers might be potent, antitumor agents.

Facilitation of parental-strain marrow engraftment by T cells of neonatally-tolerant mice.

T cells present in bone marrow cell (BMC) grafts promote engraftment in histoincompatible hosts, but they or other T cells may also initiate lethal graft-vs.-host disease (GVHD). The purpose of this study was to determine whether T cells from donors tolerant of host alloantigens were able to prevent natural killer (NK) cell-mediated rejection of BMC grafts without causing GVHD. Previous studies have shown that H2d C.B-17 SCID BMC grafts were rejected by (BALB/c x B6)F1 (CB6F1,H2d/b) host NK cells, and that this rejection was reversed by adding H2d T cells to the donor inoculum. T cells tolerant of H2d/b alloantigens were produced by irradiating (3 Gy) BALB/c newborn mice, and infusing CB6F1 BMCs. Tolerance was assessed by donor (H2b+) cell chimerism, acceptance of CB6F1 skin grafts, the inability of adoptively transferred lymphocytes to initiate GVHD in irradiated CB6F1 mice, and the inability of spleen or thymus cells to generate cytolytic T lymphocytes against H2b target cells in vitro. Whole or H2-Kb-depleted BMCs isolated from tolerant donors were able to proliferate in both BALB/c and H2b/d (C57BL/6 x DBA/2)F1 hosts as determined by incorporation of a radiolabelled DNA precursor in the spleen. Furthermore, thymocytes from tolerant donors were able to prevent rejection of H2d SCID BMCs. Because the percentage of donor F1 cells was so high in these chimeras, we generated BALB/c to CB6F1 SCID BMC chimeras; the percentage of BALB/c cells was approximately 100%, the BMCs grew well in irradiated CB6F1 hosts, and their lymph node cells failed to cause a graft-vs.-host (GVH) reaction in CB6F1 hosts. Thus, GVHD may be prevented without inhibiting the ability of donor T cells to promote engraftment. Perhaps separate T cells, or separate functions of a common T cell subset, induce GVHD and enhance engraftment of stem cells.

Combination immunotoxin treatment and chemotherapy in SCID mice with advanced, disseminated Daudi lymphoma.

We describe the use of an immunotoxin (IT) cocktail (anti-CD22- and anti-CD19-ricin A chain) and any 1 of 3 chemotherapeutic drugs (doxorubicin, cytoxan or camptothecin) to treat advanced disseminated Daudi lymphoma in SCID mice (SCID/Daudi). In a previous report, we demonstrated that this regimen was curative when given the day following tumor cell inoculation. Here, we show that combination therapy in mice with advanced tumor significantly increased their survival, although it was not curative. Importantly, the outcome of therapy was dependent upon the temporal order in which IT and chemotherapy were administered. Thus, the best anti-tumor effect was achieved when an IT cocktail was given before or at the same time as chemotherapy. When the IT was given after chemotherapy, there was no additional therapeutic benefit. Our results confirm the rationale of using combination therapy in the treatment of advanced B-cell neoplasia and suggest that ITs should be administered prior to or during chemotherapy.

Effect of sublethal irradiation of SCID mice on growth of B-cell lymphoma xenografts and on efficacy of chemotherapy and/or immunotoxin therapy.

Severe combined immune deficiency (SCID) mice obtained from different vendors vary in their capacity to accept human tumor xenografts, and in some instances mice must first be irradiated. It has been reported that SCID mice are particularly sensitive to irradiation which, in addition, can partially restore their immune system so that they are no longer totally immunologically incompetent. For the past several years we have used nonirradiated SCID mice to grow human B-cell lymphomas. When we changed vendors, we found it necessary to irradiate the mice before xenografting. Sublethal irradiation of at least one source of SCID mice before tumor cell inoculation improved tumor take and dissemination, but irradiation changed the response of these mice to chemotherapy and immunotoxins. Thus the irradiated mice did not respond to chemotherapy, but the two immunotoxins used for therapy became more effective in extending survival of the mice. It then appears that irradiation affects the immune system of SCID mice in such a way as to change their response to the therapeutic regimens used here.

P-wave duration in a clinically normal hound population.

Electrocardiogram (ECG) analysis is a common noninvasive technique used for diagnosis of cardiac disease in the clinical and research branches of veterinary medicine. Accurate analysis of P-wave duration, amplitude, and morphology is crucial to identification of morphologic and functional changes of the atria. The published accepted maximal normal value for P-wave duration in the dog is < or = 40 milliseconds. We looked at P-wave duration in ECG obtained as part of routine quarantine health screening over a period of 1 year in 364 clinically normal hounds weighing 13 to 35 kg. The dogs were neither anesthetized nor sedated and were placed in standard position. P-wave duration was classically determined from the lead-II recording. Mean P-wave duration for all dogs (44.9 +/- 6.1 milliseconds) was greater than published accepted normal values for the dog. There was a significant difference in mean P-wave duration by body weight (P < 0.001); dogs weighing > or = 20 kg had longer mean P-wave durations than dogs weighing < 20 kg (45.3 and 41.6 milliseconds respectively). There were also significant differences in mean P-wave duration by sex (P < 0.01), with a greater mean duration for females (45.4 milliseconds) than for males (43.8 milliseconds). All other ECG parameters were within published accepted normal values. A P-wave of prolonged duration leads to a diagnosis of abnormalities in cardiac morphology and/or function. Published accepted normal values for P-wave duration, at least for a clinically normal hound population, appear to be shorter than the true normal values. An error in published accepted normal standards may lead to overdiagnosis of cardiac abnormalities, as well as to erroneous results in cardiovascular studies. Therefore we recommend that the standard for P-wave duration be increased above the currently accepted standard of < or = 40 milliseconds.

Protection of mice against experimental murine mycoplasmosis by a Mycoplasma pulmonis immunogen in lysogenized Escherichia coli.

A construct of the Mycoplasma pulmonis (MP) genomic library, using randomly sheared DNA, was cloned in lambda gt11 and transfected into C600 Escherichia coli organisms. Clones of E. coli expressing a fusion protein reactive with anti-MP and monospecific serum were transferred orally or intravenously into Balb/c mice. Expression of the fusion protein was induced by adding isopropyl-beta-D-thiogalactopyranoside to the drinking water. This vaccination protocol led to local and systemic antibody formation, to generation of immune lymphocytes and to protection against large numbers of virulent MP organisms. This approach might be generally successful in preventing infectious disease.

Extra-adrenal perirenal myelolipoma.

Extra-adrenal perirenal myelolipomas are rare benign tumors that consist of adipose and hematopoietic tissue. We report a case of perirenal myelolipoma in an otherwise healthy man and review the literature.

Differential white blood cell counts as a preliminary screen for severe combined immunodeficient congenic mice.

Severe combined immunodeficient (SCID) mice are becoming increasingly popular as research animals; as a consequence, more efforts to produce congenic strains carrying the scid gene are underway. In an attempt to conserve time and resources in this endeavor, we used peripheral blood differential white blood cell counts as a preliminary screen to eliminate the homozygous (+/+) wild type and heterozygous (scid/+) animals from intercross generations. The results of our investigation confirm that blood smears can be used as a screen at four weeks of age to identify animals having an inversion of the granulocyte:mononuclear cell ratio. Mice not having an inversion of this ratio, i.e., mononuclear cells exceeding 50%, can be eliminated from the colony. This screen permits elimination of a large portion of the intercross generation one month earlier than other methods that rely on detection of serum immunoglobulin. The screen is highly sensitive and specific. We do not propose that this screen be used as a definitive test but as a tool to eliminate the majority of animals that are not homozygous at the scid locus.