The role of interferons in the control of HIV replication in macrophages.

Interferons can suppress the replication of certain retroviruses, including oncogenic murine retroviruses. In recent studies of the Lentivirinae subfamily of Retroviridae, an endogenous, immunologically induced interferon was found to restrict the replication of visna in macrophages. Several studies have shown that the replication of a human lentivirus, the human immunodeficiency virus (HIV), is also susceptible to interferon control. Here we review the evidence that interferons can protect macrophages from HIV in vitro. Macrophages treated with interferons or bacterial lipopolysaccharide (LPS) become essentially nonpermissive for HIV replication. Using the polymerase chain reaction to amplify HIV proviral DNA, we now report that interferon and LPS act to restrict the formation of proviral DNA. Effects on any several steps in the HIV life cycle may explain this data, and single-cycle infection studies are needed to define the precise roles of these agents. Taken together, these findings may explain the restricted replication of HIV in macrophages in vivo and suggest an antiviral role for endogenously produced interferon in the maintenance of the prolonged asymptomatic period which typically follows HIV infection. Interferons are currently undergoing clinical trials to determine if they have antiviral effects in HIV-infected patients.

Inpatient treatment for depressed children and adolescents: preliminary evaluations.

Reported are the results of two uncontrolled outcome studies that evaluate the effectiveness of inpatient psychiatric treatment of children and adolescents suffering from clinical depression. Study 1 employed a sample of 7 children and measured outcome with the Depression Self-rating Scale (DSRS), the Hopelessness Scale for Children (HSC), and the Global Asssessment of Functioning (GAF) scale, which were administered to each child upon admission and again at discharge. Inpatient treatment involved multiple interventions, including individual psychotherapy, medication, milieu therapy and token economy, and other procedures. At discharge, statistically significant improvements were found on the patients' GAF and HSC scores, but not on their DSRS scores. Study 2 used a sample of 15 adolescents, also admitted for clinical depression. Administered at each patient's admission and discharge, the Beck Depression Inventory (BDI), Generalized Contentment Scale (GCS), and Index of Self-esteem (ISE) were used to measure outcome. The multi-modal treatment program offered to the sample in Study 2 was similar to that offered the sample in Study 1. At discharge all three outcome measures reflected statistically significant improvements in the patients studies; therefore, these results provide addmtional support for the inpatient treatment of depressed children and adolescents.

Interferons and bacterial lipopolysaccharide protect macrophages from productive infection by human immunodeficiency virus in vitro.

To determine the effects of immunomodulatory agents upon HIV replication in macrophages, cultured monocyte-derived macrophages were treated with various substances and then infected with a macrophage-tropic strain of HIV-1. Pretreatment with rIFN-alpha, IFN-beta, and IFN-gamma, or bacterial LPS prevented viral replication in macrophages. In treated cultures, little or no infectious HIV or p24 core antigen was released into the supernatant, no virions were seen by electron microscopy, no viral RNA or DNA was detectable in the cell lysates, and no cytopathology (as determined by multinucleated giant cell formation) occurred. In contrast, pretreatment with a wide dose range of recombinant IL-1 beta, IL-2, IL-4, IL-6, M-CSF, TNF, or lymphotoxin failed to protect macrophages from productive infection by HIV. A consistent effect of granulocyte/macrophage-CSF on HIV replication in macrophages was not observed. In dose response studies, pretreatment with approximately 100 U/ml of IFN-alpha, approximately 10 U/ml of IFN-beta, or approximately 100 U/ml of IFN-gamma was sufficient to prevent virion release maximally and to prevent cytopathology completely. In kinetic studies, IFN-alpha, IFN-gamma, or LPS were added to the macrophage cultures either before or after infection with HIV. Even when added 3 d after infection with a multiplicity of 1 50% tissue-culture infectious dose per cell, all three treatments markedly reduced virion release, suggesting that these agents act at a point in the viral life cycle beyond the early events of virus binding, penetration, and uncoating. These data indicate that HIV replication in previously uninfected macrophages may be regulated by an inducible host cell mechanism. These findings may explain the restricted replication of HIV in macrophages in vivo and suggest an antiviral role for interferons in the therapy of HIV infection.

Enzyme immunofiltration staining assay for immediate diagnosis of herpes simplex virus and varicella-zoster virus directly from clinical specimens.

A simple, sensitive, 30-min assay for the detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) antigens in clinical specimens is described. The assay utilizes a filter manifold, biotinylated monoclonal antibodies, streptavidin-horseradish peroxidase conjugate, and the substrate aminoethylcarbazole. The method stains infected cells and cell debris a bright red and is easily interpreted with a dissecting microscope. Reconstruction experiments indicated that as few as two virus-infected cells per swab could be detected. This was equivalent to an infectivity titer of 29 to 160 50% tissue culture infective doses per infected cell and 250 times more sensitive than an enzyme immunofiltration assay which detects a soluble reaction product. Clinical swab specimens collected from ocular, oral, skin, and genital lesions were cultured for virus, and the remaining cell debris was immobilized on glass fiber filters and stained for the presence of virus antigens. Of 71 HSV culture-positive specimens, 66 (93%) were positive for HSV antigens. All of nine VZV culture-positive specimens were positive for VZV antigens. Of 98 culture-negative specimens, 7 were positive for either HSV (n = 3) or VZV (n = 4) antigens.

Selective binding affinity of human plasma fibronectin for the collagens I-IV.

The binding of plasma fibronectin to the collagens I, II, and III was greater in a cohort of glaucoma patients. In contrast, binding of plasma fibronectin to collagen type IV was less in 39 glaucoma patients than in any of 3 other diagnostic categories, including 92 patients that were normal, cataract, and glaucoma suspect patients. This observation may have significance in further understanding the control of aqueous outflow resistance in glaucoma and nonglaucoma patients.

Immunoenzymatic staining of viral and chlamydial antigens in cell culture.

Methods are described for the conjugation of antibodies with biotin and for the use of these reagents in an immunoperoxidase staining procedure for infected cell cultures. This technique provides a simple, rapid, and specific approach to the identification and characterization of a number of viral and chlamydial isolates in the diagnostic laboratory.

Fibronectin in human trabecular drainage channels.

Fibronectin, an extracellular glycoprotein, has been shown to be produced by human trabecular cells in culture by our group as well as Polansky and co-investigators. Studies of Rodrigues et al suggested that fibronectin may be one of several glycoproteins found in increased amounts in the corneoscleral trabecular meshwork of glaucomatous eyes. The authors have developed a sensitive immunoassay utilizing avidin-biotinylated enzyme complex (ABC) to detect low levels of fibronectin in frozen sections of human eyes. The authors have used this immunoassay together with a perfusion technique to demonstrate distribution patterns of fibronectin present in human aqueous drainage channels. The authors found that fibronectin is present in larger quantities in the aqueous drainage channels than in the surrounding tissues in 18 eyes from older patients.

Rapid viral diagnosis.

The advent of antiviral chemotherapy provides a strong impetus to develop methods to diagnose viral infections rapidly and accurately. Several other potential contributions of rapid viral diagnoses to patient management also exist. Virus isolation remains the "gold standard" of viral diagnosis against which most newly developed diagnostic approaches--including serologic testing, viral-enzyme detection, microscopic techniques, radioimmunoassays, and enzyme immunoassays--must be compared. Since, as currently performed, enzyme immunoassays such as enzyme-linked immunosorbent assays have reached the limit of their sensitivity, fully satisfactory rapid viral diagnosis will require new approaches. Two such potentially useful approaches are the detection of viral antigen with a method that permits visual localization of virus-specific immunoenzymatic staining and the detection of viral nucleic acids in clinical specimens by hybridization with nucleic-acid probes.

An enzyme immunofiltration assay useful for detecting human monoclonal antibody.

A microenzyme-linked immunoassay (EIA) utilizing an immunofiltration manifold has been developed which provides a rapid, simple, and sensitive method of detecting human monoclonal antibody class, concentration, and specificity. In this assay either whole cells or soluble antigens were immobilized on glass fiber filters followed by incubating with the test human hybridoma supernatant with subsequent analysis by EIA. A specially designed 96-chamber immunofiltration plate is employed which serves as both an incubation chamber and as a filtration manifold. The assay described is unique in that small volumes of human hybridoma supernatant are required, crude preparation of only a few target cells are needed, labile cell surface antigens are preserved and it can be completed in 3 h. This assay is well suited for the rapid screening of large numbers of human hybridoma supernatants.

Antigenic analysis of strains of Ebola virus: identification of two Ebola virus serotypes.

A sensitive radioimmunoassay has been adapted for Ebola virus antigens and antibodies to them. It uses 125I-labeled staphylococcal protein A and a specially designed filter manifold. The assay is applicable to the sera of humans and to a wide range of animal sera. Virus isolates from two discrete outbreaks of Ebola hemorrhagic fever that occurred in 1976 were shown by this assay to be antigenically distinct. This lack of identity was further confirmed by cross-absorbing antisera to each isolate with antigens of the heterologous virus strain. The advantages of this assay include the use of noninfectious antigens, the requirement for only small quantities of serum, the capability of screening large numbers of sera, the speed of execution, and the objectivity of end point determination.

Growth of cultured corneal endothelial cells onto a vitreous carbon matrix.

Fourth passage cells of a rabbit corneal endothelial line were grown for 1 week in flasks containing pieces of a reticulated vitreous carbon matrix. The rate of cell growth in flasks containing the matrix was consistent with that in control flasks. Small fragments of the vitreous carbon material lying on the flask floor were covered by the monolayers as the cells grew to confluency. Vertical growth of cells onto larger pieces of the matrix proceeded in a staged fashion with maximum cell density on pieces of the matrix closest to the floor of the flask. As defined by scanning electron microscopy, cell growth occurred to a level at least 600 microns above the floor of the flask and the confluent monolayer. This novel culture procedure should be a model situation for study of many different aspects of the in vitro capabilities of corneal endothelial cells.

Development of a rapid microELISA assay for screening hybridoma supernatants for murine monoclonal antibodies.

A micro enzyme-linked immunosorbent assay (ELISA) utilizing a filtration method has been developed which allows the rapid, simple, and sensitive detection of monoclonal antibodies that recognize either soluble or cell surface antigens. This assay involves the immobilization of target cells (or soluble antigen) onto glass fiber filter discs followed by an incubation with the test hybridoma supernatant and subsequent analysis by ELISA. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. This microELISA requires small volumes of antiserum, few target cells, and can be completed in less than 2 h. This assay is well suited for the rapid screening of murine hybridoma supernatants and can be adapted to detect monoclonal antibodies from other species.

Enzyme immunofiltration technique for rapid diagnosis of herpes simplex virus eye infections in a rabbit model.

A rapid enzyme immunofiltration assay for herpes simplex virus (HSV) has been developed which is sensitive enough to detect viral antigens in eye swabs from rabbits with primary herpes keratitis. This assay employs a specially designed filter manifold to immobilize whole cells and cell debris dissociated from the swabs. Viral antigens trapped on the filters are then detected in an indirect immunoassay utilizing staphylococcal protein A conjugated with horseradish peroxidase. The assay required only 2.5 h to perform and could be read visually. Reconstruction experiments indicated that antigen from as few as 49 HSV-infected cells could be detected. Calcium alginate swabs were shown to recover more viral antigen than dacron swabs. The enzyme immunofiltration assay detected HSV antigens on 95% of the eye swabs from which infectious virus was recovered. In addition, HSV antigen was also detected in several swabs from infected eyes which did not yield infectious virus, presumably because the virus was neutralized by native antibody present in the lacrimal fluid. This enzyme immunofiltration assay technique lends itself to the elution of native antibody bound to the viral antigens, and this may be especially applicable in the diagnosis of recurrent HSV keratitis, where antiviral antibody in the lacrimal fluid may interfere with virus isolation and fluorescent-antibody or other virus detection assays.

Fibronectin production by cultured human trabecular meshwork cells.

Postmortem explants of human trabecular meshwork from 23 eyebank eyes had a growth rate of 32% (71/160) cultures, with 8% (12) showing abundant cellular spread. One hundred percent confluency was achieved with eyes from donors of 11 to 40 years of age. One of the cultures grew profusely and we were able to study it through eight passages in culture. This culture had the morphologic characteristics described by Polansky et al. for cultured human trabecular meshwork cells. A sensitive radioimmunoassay for fibronectin in tissue culture supernatant showed an increase in fibronectin secretion over a 96 hr period, with a characteristic shift-down response occurring between 24 and 48 hr postmedia change. Three of four cultures showed the presence of laminin, a basement membrane protein, establishing that the cells are not fibroblasts.

The binding of staphylococcal protein A by the sera of different animal species.

The capacity of purified immunoglobulin or serum to bind (125I)-labeled staphylococcal protein A (SPA) was measured by means of an immunofiltration assay that facilitated the examination of large numbers of sera and required only a minute quantity of each. Sera from 80 species, including humans, laboratory animals, domestic animals, and a variety of African mammals were examined. A wide interspecies variation in the SPA-binding capacity of serum immunoglobulins was confirmed. Only small variations were observed among individuals within the same species with one notable exception. A greater than 10,000-fold variation in SPA-binding capacity was observed among sera from nine goats. Interspecies differences in serum SPA-binding capacity correlated well with taxonomic differences, and the serum SPA-binding capacity correlated well with taxonomic differences, and the serum SPA-binding capacities of African mammals corresponded closely to those of their more common relatives. The sensitivity of antibody detection by indirect (125I)SPA immunoassay was shown to be determined mostly by the SPA-binding capacity of the serum examined. The titer of antibody to influenza A/Texas/1/77(H3N2) virus in sera from several different species, when measured by (125I)SPA immunofiltration assay, was directly proportional to the SPA-binding capacity of the serum and was not proportional to the hemagglutination inhibition titer. An (125I)SPA immunofiltration assay for antibody to Lassa virus in the serum of Mastomys natalensis (which binds SPA only one-thousandth as well as human serum) was at least as sensitive as the standard fluorescent antibody assay.

Rapid serological technique for typing herpes simplex viruses.

A rapid technique is described which can accurately identify a herpes simplex virus (HSV) isolate as type 1 or type 2. Filter paper disks were used to immobilize viral antigens, which were then identified by means of an (125)I-labeled staphylococcal protein A immunoassay. The assay was performed in a specially designed 96-well filtration device which served as both an incubation chamber and a filter manifold. By using this system and cross-absorbed antisera to HSV types 1 and 2, 69 coded clinical isolates of HSV were correctly and unequivocally typed. HSV was also clearly distinguished from varicella-zoster virus and cytomegalovirus. This assay can be rapidly executed (less than 2 h) and yielded an objective endpoint; it required only minute quantities of typing sera and can be easily performed with the cells from a single infected roller tube culture. Thus, it can be used to type initial clinical isolates of HSV, yielding results within hours after the first appearance of cytopathic effects in the culture used for primary virus isolation. Moreover, it is particularly well suited to the simultaneous analysis of many specimens and is amenable to automation. These characteristics suggest that this (125)I-labeled staphylococcal protein A immunofiltration technique will be applicable to the rapid identification of other herpesviruses, as well as other clinical isolates.

Humoral and cell-mediated immunity in peripheral blood following introduction of antigen into the middle ear.

Serum levels of specific IgG and the sensitization of peripheral blood T-lymphocytes were measured in guinea pigs after single-dose antigenic sensitization by two routes: intratympanic and intradermal injection. Keyhole limpet hemocyanin (KLH) served as the antigen. Intratympanic injection of antigen resulted in much lower levels of circulating anti-KLH IgG than intradermal injection. When KLH was conjugated with alum to produce nonspecific inflammation and serve as adjuvant, the intratympanic route was considerably enhanced, but remained much less effective than the intradermal route. Development of an IgG response was also somewhat less rapid following intratympanic than following intradermal administration. Marked sensitization of circulating T-lymphocytes was seen after intradermal injection of alum-precipitated KLH. A much weaker, though still positive, response was seen after intradermal injection of KLH alone and with the intratympanic injection of alum-precipitated KLH. No T-lymphocyte sensitization could be detected after intratympanic injection of KLH alone. It was concluded that the afferent limb of both humoral (IgG) and cell-mediated immunity was operative in the middle ear. Therefore, the middle ear does not represent an immunologically "privileged" site. On the other hand, the afferent limb from the middle ear appears to operate less effectively and rapidly than that from the dermis. This observation is consistent with observations in other mucosal systems.

A rapid enzyme immunofiltration technique using monoclonal antibodies to serotype herpes simplex virus.

A rapid enzyme immunofiltration technique using monoclonal antibodies to serotype herpes simplex virus is described. It requires only a single tube culture showing viral cytopathology and can accommodate multiple specimens in a single assay. The monoclonal antibodies confer absolute specificity and the use of horseradish peroxidase-conjugated staphylococcal protein A or antiglobulin permits easy visual interpretation of the results following the 2-3 hour assay. Although it is possible that an occasional wild strain of HSV might escape recognition by monoclonal antibody, this potential problem has not been observed among the more than 500 clinical isolates tested to date, all of which have yielded an unequivocal result.

A rapid radioimmunoassay using 125I-labeled staphylococcal protein A for antibody to varicella-zoster virus.

A sensitive radioimmunoassay for serum antibody to varicella-zoster virus is described; it uses 125I-labeled staphylococcal protein A and a specially designed immunofiltration apparatus. The assay accurately distinguishes between individuals who are susceptible and those who are immune to infection with varicella-zoster virus. In addition, it can detect passive antibody in recipients of varicella-zoster immune globulin. This radioimmunoassay also detects the heterologous antibody responses that occasionally occur in patients infected with herpes simplex virus, which also have been detected by other antibody assays. The particular advantages of this assay are the use of noninfectious reagents, the speed of execution (less than 3 hr), the requirement for only small quantities of serum (30 microliters), the objectivity of end-point determination, and the capability of screening large numbers of sera. Consequently, this radioimmunoassay is especially useful for the rapid identification of susceptible individuals, which is essential for the appropriate management of patients and hospital personnel after exposure to varicella.