Inhibition of human immunodeficiency virus by N-methylisatin-beta 4':4'-diethylthiosemicarbazone and N-allylisatin-beta-4':4'-diallythiosemicarbazone.

N-methylisatin-beta 4':4'-diethylthiosemicarbazone(M-IBDET) and N-allylisatin-beta-4':4'-diallylthiosemicarbazone(A-IBDAT ) inhibit the production of Human Immunodeficiency virus (HIV). Virus inhibition was related to the thiosemicarbazone derivative (TSCD) concentrations and time of treatment. Inhibition of HIV production was confirmed by various parameters of virus assay employing reverse transcriptase activity, plaque forming units (PFU) and levels of viral structural proteins. Effective antiviral TSCD concentrations ranged from 0.17 microM to 2.04 microM for M-IBDET, and from 1.45 microM to 17.4 microM for A-IBDAT. Treatment of the chronic HIV-infected cells for 48 h with 0.34 microM M-IBDET or 2.9 microM A-IBDAT caused about 50% inhibition in as virus yield ED50 as assayed by the PFU method. Almost 2 logs of virus infectivity (PFU) was suppressed after 48 h of treatment with 17.4 microM A-IBDAT. Therapeutic index values of 20 and 30 were found for M-IBDET and A-IBDAT, respectively. A significant selective inhibition of HIV structural protein synthesis was shown by both M-IBDET and A-IBDAT.

Comparison of detection of antibody to the acquired immune deficiency syndrome virus by enzyme immunoassay, immunofluorescence, and Western blot methods.

There was 100% agreement between enzyme immunoassay (EIA) (Abbott Laboratories), Western blot, and indirect immunofluorescence (IF) when these three methods were used to measure antibody to the acquired immune deficiency syndrome (AIDS) virus in sera from 142 high-risk individuals, indicating that IF was a sensitive alternative method for detecting antibody to this agent. Thirty-two (64%) of 50 EIA-positive plasma specimens from a blood bank and 6 (21%) of 28 EIA-positive sera from alternative testing sites were negative by IF. In addition, two EIA-negative sera from the latter group were positive by IF. Western blotting agreed with IF on those 40 specimens which gave discrepant results by EIA and IF. The IF method was determined to be equal to Western blotting in sensitivity and specificity for detection of AIDS antibody, and it was found to be useful for confirming positive EIA results, especially in specimens from individuals in low-risk groups.

Anomalous antibody responses in viral infection: specific stimulation or polyclonal activation?

Eighteen paired serum samples submitted for serodiagnosis of current infection showed anomalous antibody results by complement fixation test when tested with a battery of agents (viruses, Mycoplasma pneumoniae, and chlamydia) selected for testing on the basis of the symptoms of the patient. Seventeen serum pairs showed a fourfold or greater rise in titer of antibody to two agents in the battery, and one showed only a twofold rise in titer of antibody to the identified causative agent but an eightfold rise in titer of antibody to a heterologous agent. The 18 serum pairs were tested for IgM antibody to the two involved agents to determine whether IgM antibody tests would better distinguish the probable cause of the current infection. The serum pairs were separated into three groups based on their IgM responses. Group I consisted of six serum pairs with IgM antibody to both agents, four pairs of which showed a fourfold or greater rise in titer of IgM antibody to both agents, and two of which showed a rise in titer of IgM antibody to only one of the two agents. Group II consisted of 10 serum pairs with IgM antibody to one of the two agents, 7 pairs of which showed a fourfold or greater rise in titer of IgM antibody to the agent. Group III consisted of two serum pairs with no IgM antibody to either agent. Results show that determination of presence or absence of IgM antibody per se or demonstration of a fourfold or greater rise in specific IgM antibody titer does not always help in distinguishing the causative agent in current infections.

Detection of Giardia lamblia by immunofluorescence.

High-titer immune sera to cysts of Giardia lamblia, produced in guinea pigs, were labeled with fluorescein isothiocyanate. The resulting conjugates were used to detect G. lamblia in stool specimens by fluorescence microscopy. The sera also reacted with cysts of Chilomastix mesnili, but the two organisms could be differentiated by their size.

Improved immunofluorescence antigens for detection of immunoglobulin M antibodies to Epstein-Barr viral capsid antigen and antibodies to Epstein-Barr virus nuclear antigen.

Epstein-Barr viral capsid antigen and nuclear antigen produced by modified procedures were evaluated for use in measuring viral capsid antigen immunoglobulin M and Epstein-Barr virus nuclear antigen antibody responses in sera from patients with suspected Epstein-Barr virus infections. Viral capsid antigen production was stimulated with a phorbol ester, and the Epstein-Barr virus nuclear antigen cells were fixed in suspension to eliminate loss of antigen during the drying process. Both preparations proved to be sensitive and reliable.

A 27-nm virus isolated during an outbreak of acute infectious nonbacterial gastroenteritis in a convalescent hospital: a possible new serotype.

An outbreak of acute infectious nonbacterial gastroenteritis began among elderly patients in a convalescent hospital in Marin County in northern California in March 1978 and persisted through May 1978. The overall clinical attack rate was 51% of 187 residents and 12% of 180 employees. A 27-nm viruslike particle was observed by immune electron microscopy in stools obtained at or near the onset of illness from four of 32 patients. Seroresponses to the 27-nm particles were found by immune electron microscopy in 16 of 18 patients. In addition, serologic evidence of infection with this or a related agent was demonstrated in persons who developed illness in another large outbreak of acute infectious nonbacterial gastroenteritis which occurred in a nearby county. This agent is morphologically similar to but serologically unrelated to the Norwalk and Hawaii gastroenteritis agents and has been designated the Marin agent pending further classification.

Multiple-antigen slide test for detection of immunoglobulin M antibodies in newborn and infant sera by immunofluorescence.

A microimmunofluorescence test was evaluated for use in measuring immunoglobulin M (IgM) antibodies in infant sera to five of the agents implicated in congenital and neonatal disease. Pen point dots of Toxoplasma gondii, cytomegalovirus, rubella virus, herpes simplex virus, and chlamydial cell culture antigens were applied to each circle of eight-circle printed slides. These multiple-antigen slides greatly facilitated the screening of 607 sera from infants and 117 sera from mothers for the presence of IgM antibody to these agents. Forty sera could be examined microscopically in approximately 30 min. All sera reacting with one or more antigens were tested for rheumatoid factor by the latex method, absorbed with glutaraldehyde-cross-linked human IgG, and retested for the presence of IgM antibody. IgM antibody to cytomegalovirus was demonstrated in sera from four newborns, but IgM antibody to rubella virus could not be detected until 21 days after birth, although rubella virus was isolated from sera from five younger infants. This may indicate that rubella IgM levels in many congenitally infected newborns are too low to be measured by the immunofluorescence method. Five percent of the sera from infants in this study possessed demonstrable IgM antibody to one of the antigens.

Differentiation of cytomegalovirus antigens by their reactivity with various classes of human antibodies in the indirect fluorescent antibody test.

Human sera containing immunoglobulin G (IgG), IgA, and IgM antibodies were tested by immunofluorescence for reactivity with cytomegalovirus-infected cell cultures and with early antigens of cytomegalovirus produced by treating the infected cultures with either bromodeoxyuridine or cytosine arabinoside. IgG antibody but not IgM antibody reacted with early antigens produced in bromodeoxyuridine-treated infected cultures. This observation on a small sample of sera suggested that a positive IgM reaction with an infected, nontreated culture and a negative reaction with a bromodeoxyuridine-treated infected culture may indicate a positive specific IgM reaction for cytomegalovirus, even in the presence of IgM rhematoid factor. The hyothesis requires further testing. The different classes of antibody did not all react or did not react to the same extent with early antigens produced in infected cells blocked with cytosine arabinoside or bromodeoxyuridine. This observation indicates that different antigens were being produced as a result of the two treatments.

Production of fibronectin by human epithelial cells in culture.

Human epithelial cell lines derived from both carcinomatous and nomalignant tissues were characterized with respect to the presence and distribution of fibronectin by immunofluorescence microscopy. In cell lines derived from nonmalignant tissues or from primary carcinomas, fibronectin was found predominantly in an extracellular matrix. In contrast, cell lines derived from metastatic carcinomas displayed very little or no fibronectin. Metabolic labeling studies indicated that a positive line synthesized fibronectin de novo rather than absorbing the protein from the media. Negative lines neither synthesized fibronectin nor secreted it into the culture fluid, suggesting that they were not producing fibronectin. Evidence is presented that cells in culture change their properties after extensive subculture since a small amount of fibronectin in an extracellular matrix was observed after extensive subculture of two metastatic lines that were originally negative.

Properties of epithelial cells cultured from human carcinomas and nonmalignant tissues.

Human epithelial cell cultures were examined for expression of plasminogen activator and fibronectin matrix. All of the cells examined showed ultrastructural evidence suggesting their epithelial origin, including microvilli and specialized junctions. The nonmalignant cells were also negative for endothelial cell markers (ie. they lacked factor VIII antigen, a nonthrombogenic surface and Weibel-Palade bodies). The nonmalignant lines all produced large amounts of plasminogen activator, whereas the tumor-derived lines showed a gradation of activities, ranging from lines having as much activity as the nonmalignant lines to lines having little or no activity above background. For both normal and malignant cells, addition of dexamethesone only slightly decreased the levels of plasminogen activator. By immunofluorescence microscopy, normal bladder and fetal intestine epithelial cells showed fibronectin in a globular and fibrillar matrix. In contrast, normal mammary epithelial cells had a much diminished amount of fibronectin with a punctate distribution.

Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and fecal samples.

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO(2) incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.

Photochemical inactivation of DNA and RNA viruses by psoralen derivatives.

Western equine encephalitis virus, and RNA virus, and herpes simplex virus type I, a DNA virus, were efficiently inactivated in less than I min by exposure to long-wave ultraviolet light (320 to 380 nm) in the presence of several psoralen derivatives. The psoralen photochemical reaction was chosen for study due to its known specificity for nucleic acids. Neither the light nor any of the drugs alone caused appreciable inactivation. The inactivation kinetics and dependence on light intensity and on different derivatives of psoralen were studied. The high solubility of a new aminomethyl psoralen derivative was found to be advantageous in the photochemical inactivation of the RNA virus, but was not in the case of the more easily inactivated DNA virus. Within its limited solubility range trimethylpsoralen was superior to its aminomethyl derivative on a molar basis for the inactivation of both types of viruses under most of the conditions studied.

Two syngeneic cell lines from human breast tissue: the aneuploid mammary epithelial (Hs578T) and the diploid myoepithelial (Hs578Bst) cell lines.

We characterized two human cell lines (Hs578T and Hs578Bst), which provide several unique features that should be useful in the study of breast disease. Hs578T, derived from a carcinosarcoma, is epithelial in origin. Hs578Bst, established from normal tissue peripheral to the tumor, is myoepithelial in origin. This is the first report of companion cell lines, one malignant and one normal, established from the same organ.

Distinction between envelope antigens of murine xenotropic and ecotropic type C viruses by immunoelectron microscopy.

The indirect ferritin-labelled antibody technique was used to determine the reactivity of an antiserum prepared against the NZB xenotropic virus with three murine xenotropic viruses, a feline xenotropic virus and a murine ecotropic virus. The envelope antigens of the xenotropic type C viruses isolated from the NZB, NIH Swiss and C57L mice were tagged with ferritin. The feline RD114 virus was not. Gross murine leukaemia virus was tagged, but only at high serum concentrations. The cross-reactivity titre of Gross virus to anti-NZB serum was removed by a serum dilution which was still reactive to xenotropic viruses. This difference in reactivity titres between a xenotropic and an ecotropic virus was sufficient to distinguish one from the other in doubly infected cultures. Specific tagging of membrances of cells infected by xenotropic virus was also observed.

Expression of antigenic crossreactivity to RD114 p 30 protein in a human fibrosarcoma cell line.

An antigen crossreacting with the 30,000-molecular-weight protein (p30) of the feline endogenous oncornavirus (RD114) was detected in a well-characterized human fibrosarcoma cell line, HT1080, by indirect immunofluorescence. Three antisera against RD114 p30 gave similar positive results, while two antisera prepared against simian sarcoma virus p30, one antiserum prepared against murine leukemia virus p30, and one antiserum prepared against feline leukemia virus p30 gave no immunofluorescence. The reactivity observed with the antiserum against RD114 p30 was detected in 10-40% of the cells at early passages and was no longer expressed by the forty-first subculture. The reactivity could be removed by adsorption of the antiserum with RD114-infected dog or human cells, but not by uninfected cells or by cells infected with an antigenically unrelated oncornavirus, feline leukemia virus. Neither complete virus particles nor reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity was detected in the culture. These experiments suggest that the fibrosarcoma cell line is expressing an antigen related to the p30 protein of RD114 baboon endogenous virus group of oncornaviruses without producing complete virions.

Induction of lymphoma and associated xenotropic type C virus in C57L mice by whole-body irradiation.

Adult C57L mice received sublethal whole-body X-irradiation. Between 3 and 11 months later, 5 of the 7 exposed mice developed histopathologically confirmed malignant lymphomas (lymphocytic type) that primarily involved the thymus. The lymphomas were readily transplantable to other C57L mice of any age, which developed fetal lymphomatous involvement; the tumor cells could also be propagated in tissue culture. A xenotropic murine type C virus (MuX) was isolated from the cultured lymphoma cells after cocultivation with a permissive dog line. MuX activity was demonstrated by electron microscopy, complement fixation, indirect fluorescent antibody, infectivity, and genome rescue.

Continuing search for the etiology of cat scratch disease.

Fifty cat scratch disease patients were tested for antibodies against candidate etiological agents, with inconclusive results. Future research should emphasize new viral isolation methods to discover the elusive agent.

Viral identification by scanning electron microscopy of preparations stained with fluorescein-labeled antibody.

A new technique combines the specificity of fluorescent-antibody labeling and the resolution of the scanning electron microscope to identify and distinguish between viruses. Hemagglutination of chicken erythrocytes by influenza virus was used as a model system to demonstrate the technique.