Effect of eltenac in horses with induced endotoxaemia.

Ten horses were used in a crossover study to evaluate the effectiveness of eltenac against endotoxaemia. Eltenac (0.5 mg/kg bwt) or saline control was given i.v. then 15 min later, intravenous infusion of endotoxin was begun and continued for 120 min (total dose 100 ng/kg bwt). Horses were monitored for heart and respiratory rates, pulmonary and carotid arterial pressure and core body temperature. Blood was sampled at intervals for measurement of haematological variables and plasma concentrations of lactate, prostanoid metabolites, tumour necrosis factor (TNF) and stress hormones. In comparison with saline-treatment, use of eltenac significantly protected against endotoxin-induced changes in respiratory rate, core temperature, systemic arterial blood pressure (SAP), pulmonary arterial pressure, PCV, and plasma protein, 6-keto prostaglandin F1 alpha, thromboxane B2, epinephrine, and cortisol concentrations. Despite statistical effect of eltenac on SAP, values in both treatment groups remained well above baseline throughout the evaluation period. Significant protective effect of eltenac was not found for heart rate, white blood cell count, plasma lactate concentration or TNF activity. On the basis of these results, it is expected that use of eltenac will provide clinical benefit in horses with naturally occurring endotoxaemia.

Influenza A2 inhibits murine in vitro antibody synthesis.

A2 influenza inhibited in a dose-dependent fashion the in vitro antibody-forming cell (AFC) response of CBA mouse spleen cells to sheep erythrocytes. The greatest amount of inhibition occurred if virus was added to the splenocytes on the day the cells were placed in culture. Inoculation of spleen cells with influenza on day 3 of culture or immediately before assay on day 4 had no effect on the AFC response. Experiments indicated that the inhibition could be transferred by adding viable, infected cells to uninfected spleen cultures. The origin of the cells that caused the suppression of AFC was not resolved; however, the data suggest that infected B or T cells were not solely responsible for the inhibition. Although infectious virus was most effective, inactivated influenza preparations also suppressed the AFC response. The possible in vivo significance of influenza's inhibitory effect on antibody synthesis is discussed.

Scanning and transmission electron microscopic studies of complement-mediated lysis and antibody-dependent cell-mediated cytolysis of herpes simplex virus-infected human fibroblasts.

The morphologic aspects of complement-mediated and antibody-dependent cell-mediated cytolysis (ADCC) of human fibroblasts (HuFs) infected by herpes simplex virus (HSV) is described. Human antiviral antibody (antiHSV) was shown by transmission and scanning electron microscopy (TEM and SEM) to cause the deposition of an amorphous material over the surface of infected cells and virus particles. Associated with antiHSV treatment, the HuFs underwent endocytosis, with the appearance of pinocytotic vesicles immediately beneath the plasma membrane. The addition of complement resulted in lysis of the infected HuFs and massive dilatation of the perinuclear cisternae, but the virus particles associated with the cell surface did not appear lysed. Instead, an additional deposit was noted on the enveloped particles after the addition of complement (C). Human peripheral blood mononuclear leukocytes (MNLs) also lysed the antibody-coated, infected HuFs. Lymphocytes formed broad-based areas of attachment to the antiHSV-treated cells. Beneath these areas of contact occurred focal cytoplasmic changes that preceded cell lysis. Monocytes showed multiple points of binding and sent cytoplasmic projections over the surface of the infected HuFs. Virus particles and segments of target cell cytoplasm were gathered into vacuoles of the monocyte. In accord with the above morphologic findings, the relative roles that antibody, C, and leukocytes may play in human viral diseases is discussed.

Effect of prostaglandins and cyclic adenosine 3',5'-monophosphate modulators on herpes simplex virus growth and interferon response in human cells.

Mechanisms whereby prostaglandins and other cyclic adenosine 3',5'-monophosphate (cAMP) modulators might enhance the growth of herpes simplex virus (HSV) in human skin fibroblasts were explored. Prostaglandins A1, B1, E1, E2, and F2 alpha, as well as isoproterenol, imidazole, carbamylcholine, and dibutyryl cAMP had no effect on HSV growth. On the other hand, the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine and theophylline delayed the growth, suppressed the cell-to-cell spread, but inhibited neither the adsorption nor the penetration of the virus. Although none of the cAMP-elevating reagents directly enhanced HSV growth, they were found to inhibit dose dependently the antiviral action of both type I and HSV antigen-induced human interferon preparations. Furthermore, these reagents suppressed the production of HSV antigen-induced interferon by immune human mononuclear leukocytes. These data support the hypothesis that prostaglandin elaboration in vivo could contribute to exacerbations of HSV infections by compromising the host's interferon defense system.

Enhanced antiglobulin-mediated neutralization of herpes simplex virus-IgG complexes by complement and heterologous anti-immunoglobulin.

The ability of IgG anti-Fc and anti-Fab to neutralize infectious herpes simplex virus-IgG (HSV-IgG) complexes was determined. When limiting amounts of antiglobulin were used, antibody directed against the Fab portion of human IgG was significantly more effective than anti-Fc antibodies in neutralizing the HSV-IgG complexes. The detection of viral bound antibody was enhanced by the incorporation of heterologous antiglobulin or complement in the antiglobulin neutralization test. Specifically, HSV-IgG which had been incubated with rabbit antihuman globulin was further neutralized by goat antirabbit IgG or guinea pig serum complement. This augmented neutralization test could prove useful in detecting small amounts of antibody bound to virus in infectious isolates from patients or experimental animals with viral diseases.

Growth of type 2 herpes simplex virus in newborn and adult mononuclear leukocytes.

Growth of type 2 herpes simplex virus (HSV) in newborn and adult human mononuclear leukocytes (MNL) was compared. Phytohemagglutinin stimulation of cultures for 3 days yielded comparable peak titers in newborn (10(5.3) PFU) and adult (10(5.1) PFU) MNL. Unexpectedly, 3-day cultures of unstimulated newborn MNL also substantially replicated HSV (10(4.7) PFU), whereas similarly treated unstimulated adult cells did not. Growth of HSV in freshly isolated human MNL was next investigated. MNL from 4 mothers and 6 nonpregnant adults showed no evidence of virus growth; however, leukocytes from 11 of 24 newborns (46%) supported replication. Newborn MNL manifested an increased ability to replicate HSV within 1 day of culture, whereas comparable growth in adult MNL was not achieved until the 4th day of culture. The significance of the above observations as it relates to visceral dissemination of HSV in the neonate is discussed.

The role of protein A in the attachment of staphylococci to influenza-infected cells.

The adherence of staphylococcal protein A-containing Cowan I bacteria to influenza-infected cells was enhanced up to 5 times following incubation of the monolayers with antiinfluenzal serum, but not following treatment with nomimmune serum. Significantly increased binding of Cowan I bacteria was detected at antiinfluenzal serum dilutions as high as 1:40,960. None of the several antibody concentrations tested enhanced the binding of staphylococcal protein A-negative staphylococci. In addition, extracellular staphylococcal protein A was found to inhibit complement-mediated cytolysis of antibody-coated, virus-infected cells. The possible significance of staphylococcal protein A in the synergistic interaction between staphylococcal and influenza virus infections is discussed.

Abortive and productive infections of human mononuclear phagocytes by type I herpes simplex virus.

The ability of Type I herpes simplex (HSV) to replicate in normal human monouclear phagocytes was investigated. Mononuclear leukocytes were obtained from the peripheral blood of patients by Ficoll-Hypaque gradient centrifugation, and the monocytes were isolated by allowing the cells to adhere to tissue culture dishes. The monocytes (10(5.0) cells) were infected (10(7.0) PFU HSV) either immediately after isolation or were cultured in vitro for varying numbers of days and were then infected. Inoculation of freshly isolated monocytes resulted primarily in an abortive infection. HSV antigens were produced by the cells, as determined by a indirect fluorescent antibody technique, and empty herpes capsid structures were detected by electron microscopy of the inoculated monocytes; however, no increase in virus titer was noted in the cultures. Inoculation of viable cells that had been maintained for 7 days in culture resulted in a productive infection. An increase in titer was noted 24 hours after inoculation, and normal virus maturation was documented by ultrastructural study of the infected cells. The experiments show that the interaction of HSV with human mononuclear phagocytes is complex, and the data suggest that whether or not the cell replicates infectious virus may depend on the functional activity of the cell.

The effect of prednisolone on antibody-dependent cell-mediated cytotoxicity and the growth of type I herpes simplex virus in human cells.

Treatment of human skin and corneal fibroblasts with prednisolone-21-phosphate did not increase the capacity of these cells to replicate type I herpes simplex virus (HSV). The steroid however was found to (1) inhibit human lymphocytes from mediating antibody-dependent cell-mediated cytotoxicity (ADCC) against HSV-infected fibroblasts and (2) suppress the replication of virus in PHA-stimulated human lymphocytes. The data suggest that the exacerbation observed when patients with dendritic keratitis are inadvertently treated with prednisolone may be due to the steroid suppressing ADCC and not by promoting the growth of virus in the corneal cells.

Replication of type I herpes simplex virus in primary cultures of hairy cell leukemic leukocytes.

The ability of leukemic leukocytes to support the replication of herpes simplex virus (HSV) was studied. Mononuclear leukocytes (MNL) from the peripheral blood of patients with a variety of lymphoid leukemias were isolated on Ficoll-Hypaque gradients and infected with HSV at a multiplicity of infection of 5 to 10. No virus growth was detected in cells from patients with chronic lymphocytic leukemia (9), acute lymphocytic leukemia (1), or lymphosarcoma cell leukemia (2), HSV replication did occur in hairy cell leukemic MNL from all of 4 patients studied. Maximal titers of 10(3.7) to 10(4.7) PFU/ml occurred 1 to 7 days after incubation. By electron microscopy, herpesvirus particles were seen in the nuclei of these infected cells after 3 days of culture, but none was seen in the cells not exposed to virus. Fluorescent antibody examination confirmed the presence of HSV antigens in the nuclei of infected hairy cells. No difference in the adsorption or penetration of the virus was found with the various MNL studied. Productive infection of the cells thus appeared to depend on the ability of the leukocyte ;o support a later stage of infection, either uncoating or replication of the virus.

Effect of rheumatoid factor and anti-immunoglobulin G antibodies on complement-mediated lysis of herpes simplex virus-infected human fibroblasts.

The effect of various anti-immunoglobulin G (IgG) antibodies on the complement-mediated lysis of herpes simplex virus-infected human fibroblasts was determined. IgM rheumatoid factor, a naturally occurring anti-human Fc, inhibited lysis, whereas rabbit anti-human IgG serum potentiated immune cytolysis. We attempted to explain this disparity by determining the effect various classess of anti-IgG's with differing specificities had on complement-mediated lysis. Inhibition of cytolysis occurred with IgM anti-Fc and all of the IgG antiglobulins (anti-IgG, Fab, and Fc). In contrast, IgM anti-Fab enhanced lysis. IgM anti-IgG suppressed immune cytolysis when high concentrations of antiviral serum were incubated with the virus-infected cell, but augmented lysis when low concentrations of anti-herpes simplex virus antibody were exposed to the fibroblasts. The experiments indicated that whether a particular antiglobulin potentiates or inhibits lysis depends on the concentration of antibody bound to the target cells as well as the class and specificity of the antiglobulin exposed to the antibody-coated cell.

Effect of virus infection on the inflammatory response. Depression of macrophage accumulation in influenza-infected mice.

To better define the mechanisms by which viruses depress immune function, the effect of influenza infection on the ability of macrophages to accumulate at sites of inflammation was determined. Mice were inoculated with virus, and their inflammatory response measured in vivo by counting the number of leukocytes which accumulated in the peritoneal cavity 2 days after an intraperitoneal injection of phytohemagglutinin. Mice infected with influenza had a 57% and 65% depression of total leukocyte and macrophage accumulation, respectively, as compared to the response of uninfected mice. In contrast, bacterial pneumonia did not produce a decrease in the macrophage response. This indicated that the depression was produced by the virus infection rather than being a nonspecific phenomenon accompanying any inflammatory focus in the lung. The in vitro chemotactic responsiveness of normal peritoneal macrophages incubated with infectious influenza virus was 53% of normal. These experiments suggest that influenza infection may depress a host's ability to mobilize macrophages to inflammatory sites in vivo by inhibiting their chemotactic responsiveness.

Inhibition by rheumatoid factor, anti-FC, and staphylococcal protein A of antibody-dependent cell-mediated cytolysis against Herpes simplex virus-infected cells.

Incubation of herpes simplex virus-infected human fibroblasts with the serum from a patient with herpes labialis rendered the cells susceptible to immune lysis by human mononuclear leukocytes (MNL) as well as complement. If, before the addition of MNL, the antibody-treated, infected monolayers were incubated with either IgM rheumatoid factor (RF), staphylococcal protein A (SPA), or anti-Fc gamma serum, antibody-dependent cell-mediated cytolysis (ADCC) was markedly depressed. SPA and anti-Fc caused maximal inhibition (greater than 90%), whereas RF resulted in a 72% depression. The inhibition of ADCC was dependent on both the concentration of the Fc-reacting materials incubated with the antibody-coated target cells and the concentration of antiviral antibody incubated with the virus-infected fibroblasts. Experiments indicated that the Fc-reacting materials depressed ADCC at the target cell level by covering or altering Fc sites on cell-bound antiviral antibody.

Cytomegalovirus replication and the host immune response.

Cytomegalovirus (CMV) is closely associated with host cellular structures, and this has a significant impact upon the immunologic response following infection. CMV may be recovered from a variety of body secretions and fluids during acute infection, and protracted shedding may supervene in some instances. The reasons for a variable host response to CMV infection remain unclear, and the mechanisms responsible for the establishment of persistence have not been worked out. CMV persistence and latency are discussed, and some recently derived relevant data are presented. An animal model has been developed consistent with clinical observations pertaining to CMV transmission with blood. Results obtained in the course of these and other studies support the concept of immunological activation of latent CMV. The timing of CMV infection relative to an unrelated antigenic challenge is probably critical in determining the emergence of immunodepression or enhancement. Some aspects of CMV sero-diagnosis are also reviewed.

Depressed monocyte chemotaxis during acute influenza infection.

The chemotactic responsiveness of monocytes from patients with serologically proven influenza infection has been quantified in vitro. Individuals with acute influenza had a significant (P less than 0-001) depression of monocyte chemotaxis. The depression ranged from 40% to 72% during acute infection but rose to normal by three weeks after recovery. When isolated mononuclear leucocytes from the recovered patients were incubated with the infecting strain of virus (Port Chalmers), a 49-54% inhibition of chemotaxis was obtained. These findings support the hypothesis that the altered immune responsiveness and increased predisposition to superinfections found frequently in patients with influenza can be due to the ability of the virus to depress monocyte function.

Effect of staphylococcal protein A on complement-potentiated neutralization of herpes simplex virus and immune lysis of virus-infected cells.

Interaction of staphylococcal protein A (SPA) with human serum depressed the ability of such serum to neutralize herpes simpled virus (HSV)-antibody labialis. SPA-induced depression of serum-dependent virus neutralization appeared to be due to consumption of complement by SPA. In addition, SPA attached to antibody-treated, HSV-infected cells and inhibited complement mediated immune cytolysis. The amount of inhibition obtained depended upon the with the infected cells. The possible significance of SPA in the pathogenesis of viral disease complicated by secondary staphylococcal infection is discussed.

Shedding of infectious virus/antibody complexes from vesicular lesions of patients with recurrent herpes labialis.

The concentration of herpes-simplex virus (H.S.V.) in the lesions of adults with recurrent herpes labialis was determined. On the 1st day the vesicle appeared, the fluid within the lesion contained 10(5-3) plaque-forming units (P.F.U.) of H.S.V./mul. By swabbing the surface of the lesions with a sterile pledget, 10(6-2) P.F.U. of virus was isolated from the inflamed labial mucosa. The amount of virus obtained from the labial surface decreased on the 2nd and 3rd day to 10(5-0) and 10(3-0) P.F.U., respectively. In two patients on immunosuppressive drugs, high concentrations of virus (greater than 10(4-0) P.F.U.) were obtained per swab for more than 3 weeks. The presence of infectious virus-antibody (V.A.) complexes in herpetic lesions was demonstrated by examining fifty-two isolates from twenty-eight patients at various times during the course of their disease. 71% of the patients had V.A. complexes in their lesions on the 1st day of the vesicular eruption, and by the 3rd day all of the lesions examined contained complexes. It is concluded that patients with active lesions shed high concentrations of virus and that natural infection may be transmitted by an infectious V.A. complex.