Search

OBJECTIVES: This study compared the prevalence of health behaviors among lesbians and in the general population of women. METHODS: We used a cross-sectional community-based survey of 1010 self-identified lesbians 18 years or older. RESULTS: Compared with the general population of women, lesbians were more likely to report cigarette use, alcohol use, and heavy alcohol use. A higher percentage of lesbians were categorized as overweight, and lesbians were more likely to participate in vigorous physical activity. They were less likely to report having had a Papanicolaou test within the past 2 years but more likely to report ever having had a mammogram. CONCLUSIONS: While there may be differences in health behaviors between lesbians and the general population of women, how these differences influence the risk of subsequent disease is unknown. [ABSTRACT FROM AUTHOR]

Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddA tox , that can act upon double-stranded DNA. Here, we target DddA tox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddA tox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity., (© 2024. The Author(s).)

Further studies aimed at examining the activity of different Cu(II)-ligand complexes to serve as electron-transfer mediators to prepare novel antimicrobial/thromboresistant nitric oxide (NO)-releasing intravenous catheters are reported. In these devices, the NO release can be modulated by applying different potentials or currents to reduce the Cu(II)-complexes to Cu(I) species which then reduce nitrite ions into NO (g) within a lumen of the catheter. Four different ligands are compared with respect to NO generation efficiency and stability over time using both single- and dual-lumen silicone rubber catheters: N-propanoate- N, N-bis(2-pyridylethyl)amine (BEPA-Pr), N-propanoate- N, N-bis(2-pyridylmethyl)amine (BMPA-Pr), 1,4,7-trimethyl-1,4,7-triazacyclononane (Me 3 TACN), and tris(2-pyridylmethyl)amine (TPMA). Of these, the Cu(II)BEPA-Pr and Cu(II)Me 3 TACN complexes provide biomedically useful NO fluxes from the surface of the catheters, >2 × 10 -10 mol·min -1 ·cm -2 , under conditions mimicking the bloodstream environment. Cu(II)Me 3 TACN exhibits the best stability over time with a steady and continuous NO release observed for 8 d under a nitrogen atmosphere. Antimicrobial experiments conducted over 5 d with NO-releasing catheters turned "on" electrochemically for only 3 or 6 h each day revealed >2 logarithmic units in reduction of bacterial biofilm attached to the catheter surfaces. The use of optimal Cu(II)-ligand complexes within a lumen reservoir along with high levels of nitrite ions can potentially provide an effective method of preventing/decreasing the rate of infections caused by intravascular catheters.

Nitric oxide (NO) levels in exhaled breath are a non-invasive marker that can be used to diagnose various respiratory diseases and monitor a patient's response to given therapies. A portable and inexpensive device that can enable selective NO concentration measurements in exhaled breath samples is needed. Herein, the performance of an amperometric Pt-Nafion-based gas phase sensor for detection of NO in exhaled human nasal breath is examined. Enhanced selectivity over carbon monoxide and ammonia is achieved via an in-line zinc oxide-based filter. Exhaled nasal NO levels measured in 21 human samples with the sensor are shown to correlate well with those obtained using a chemiluminescence reference method (R 2 = 0.9836).

In recent years the Eye Department at Aalborg University Hospital has had many referrals of older children with severe amblyopia due to anisometropia. Anisometropia is not visible, thus a monocular vision examination is needed in order to detect it. Early detection increases the possibilities for a better post treatment visual acuity. Recent studies indicate positive treatment response also in older children, and some older individuals respond dramatically. Currently it is not possible to predict who will respond, and therefore treatment should be offered despite the age of the child if the child and parents are motivated for it.

Hit-to-lead medicinal chemistry efforts are described starting from a screening hit 1, leading to a new class of aryl sulfonamide-based MR antagonist, exemplified by 17, that possesses favourable MR binding affinity, selectivity profile against closely related NHRs, physicochemical properties and metabolic stability., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

By competition neutralization assay using monoclonal antibodies (MAbs) to varicella-zoster virus (VZV) glycoproteins (gps), we attempted to determine the topographical relationship of epitopes which are functional in VZV neutralization. MAbs against gpI interfered moderately to strongly with neutralization of MAbs against gpIII, and one antigenic domain with two distinct epitopes was identified on gpIII. Competition neutralization assays performed with MAbs to gpI revealed at least three distinct antigenic domains: the first contained two complement-dependent neutralizing epitopes; the second contained five complement-dependent neutralizing, overlapping epitopes and one nonneutralizing, nonoverlapping epitope; and the third contained one complement-enhanced neutralizing epitope. Competition neutralization assays performed with MAbs to gpIV showed one antigenic domain with two distinct epitopes which competed with nonneutralizing gpI MAbs. gpII did not interfere with neutralization of gpI, gpIII, or gpIV. Our data suggest that neutralizing and nonneutralizing MAbs can interfere with the action of viral neutralization either by inhibition or by enhancement. This report describes the epitope mapping of VZV gps by a functional biological assay.

This paper describes the preparation of seven combination pools of equine antisera, designated J though P, for identification of 19 coxsackievirus A immunotypes. Each pool is composed of 4 to 6 antisera; the serotypes included are A1-6, 8, 10-15, and 17-22. These pools, unlike the previously prepared A-H enterovirus pools, were lyophilized from volumes of 0.5 ml dispensed into 5-ml vials, and when rehydrated with 5 ml of diluent provide 50-antibody-unit material ready for use in identification tests without further dilution. Procedures for using the antiserum pools are given, and guidance is provided for interpreting the results of serum neutralization tests in identifying field isolates.

A peculiar cytotoxic effect, occasionally encountered in the course of inoculating cell cultures with fecal specimens for routine enterovirus isolation attempts, was shown to be produced by Clostridium difficile toxin.

An enzyme immunoassay utilizing a fluorogenic substrate (EIFA) was employed to compare humoral antibody responses to purified virion and dense body antigens in human cytomegalovirus (CMV) infections. Results of these antibody assays were also compared to those obtained by plaque reduction neutralization and complement fixation (CF). In adults with CMV infections diagnosed by a significant rise in CF antibody titer, there was also a vigorous antibody response to the virion and dense body antigens. There was better correlation between antibody levels to the virion and dense body antigens than between antibody levels detected by other combinations of tests. Antibody responses to the virion and dense body antigens were similar, but not identical, suggesting that although the two structures share major antigens, they may also possess unique antigens to which the host can mount independent antibody responses. All individuals with CMV neutralizing antibody also had virion and dense body antibodies, but there was poor correlation between neutralization titers and those detected by EIFA, perhaps reflecting greater strain specificity of the neutralization test. Some individuals with serological diagnoses of Mycoplasma pneumoniae or influenza A infection showed fourfold or greater antibody titer rises to CMV in two or more assays, suggesting reactivation of latent CMV in these infections.

Efforts were made to shorten the time required for detection of rubella virus in clinical materials through the use of immunoperoxidase (IP) staining. Comparative studies were performed in which specimens were inoculated in parallel into BHK-21 hamster kidney cells, which were examined by IP staining at 5 days, and into BK-13 and BS-C-1 cells, which were examined in two ways, viz., by subpassage at 7 days into BHK-21 cells and IP staining 3 days later and by subpassage at 7 days into BS-C-1 cells followed by interference testing and immunofluorescence (IF) staining on positive materials (standard method). Direct inoculation into BHK-21 cells with IP staining at 5 days permitted detection and identification of 59% of the 63 positive specimens. Toxicity of some specimens preserved with sorbitol and of certain tissue specimens reduced the number of satisfactory examinations which could be performed in this system. Virus detection and identification by IP staining on subpassaged RK-13 and BS-C-1 materials, requiring a total of 18 days, was comparable to the longer interference-IF method, requiring 17 days. Results obtained by IP staining and interference-IF showed 98% correlation on RK-13 materials and 97% correlation on BS-C-1 materials. IP staining on inoculated BHK-21 cells can be a useful method for rapid identification of a relatively high proportion of rubella-positive specimens, particularly if sorbitol-preserved specimens are avoided, and IP staining on subpassaged RK-13 and BS-C-1 materials is a highly satisfactory alternative to the longer interference-IF method.

Indirect immunoperoxidase (IP) staining was evaluated for sensitivity and specificity in detecting Colorado tick fever (CTF) virus antigen in infected cell cultures and infected mouse tissues, and then was applied to a study of congenital CTF infection in mice. The sensitivity of IP staining was comparable to that of immunofluorescence staining in detecting CTF antigen in infected cell cultures. Endogenous peroxidase activity of mouse tissues caused nonspecific reactivity in the IP system, but this could be abolished by treatment with sodium azide and hydrogen peroxide without destroying CTF antigen. Offspring of mice infected with CTF virus during the 2nd week of pregnancy showed a highly significant increase in the incidence of stillbirths and neonatal deaths as compared with offspring of uninfected controls. CTF antigen or virus was demonstrable in only a low proportion (7%) of embryos, ill newborns or stillborns examined, but a high proportion of mice examined at a time when maternal antibody would be lost (6 and 12 weeks) showed CTF antibody, indicating a higher incidence of infection. IP staining showed potential for use in studies of viral pathogenesis in the mouse model.

Pooled monoclonal antibodies to varicella-zoster virus (VZV) were used as "detector" antibodies in a four-phase enzyme immunofluorescence assay for determination of immunoglobulin M (IgM), IgA, and IgG antibodies to VZV. Polyclonal antisera specific for heavy chains of human IgM, IgA, and IgG were employed as "capture" antibodies on the solid phase. The antibody class capture assay (ACCA) for VZV IgM antibody detected high titers of virus-specific IgM in all patients with varicella and in 5 of 10 zoster patients. VZV IgM antibody was not detected in patients with primary herpes simplex virus infections or in other individuals without active VZV infection, with one exception, a patient with encephalitis who had other serological findings compatible with a reactivated VZV infection. VZV-specific IgA and IgG antibody titers demonstrable by ACCA were compared with those measured by solid-phase indirect enzyme immunofluorescence assay (EIFA). VZV IgA antibody titers detected in patients with varicella and zoster were variable and could not be considered to be reliable markers of active VZV infection. IgA antibody titers detected by ACCA tended to be higher than those demonstrated by solid-phase indirect EIFA in varicella and zoster patients. VZV IgG antibody titers detected by ACCA in patients with varicella, and to a lesser extent in zoster patients, were as high as or higher than those demonstrated by solid-phase indirect EIFA. However, ACCA was totally insensitive in detecting VZV IgG antibody in individuals with past infections with VZV and would not be a suitable approach for determination of immunity status to VZV.

N:NIH(S) II nu/nu (athymic) and +/nu (euthymic) mice were inoculated with coxsackievirus B3 (CBV-3) and examined at various times after infection for virus titres in the heart, myocarditis and serum neutralizing antibodies. Virus was recovered from the hearts of nu/nu mice for up to 94 days post-inoculation, but was not recovered from the hearts of any +/nu mice beyond 14 days. Inflammatory cell infiltration and necrosis were present in the hearts of +/nu mice at all harvest times (7, 14, 21 and 28 days). Inflammation and necrosis did not become evident in nu/nu mice until 14 days post-inoculation, and was the present in mice from each harvest until the end of the experiment (94 days). In athymic mice, myocarditis showed a strong correlation with persistence of CBV-3 in the heart. In N:NIH(S) II mice, the presence (+/nu) or absence (nu/nu) of a thymus had a major influence on the clearance of virus from the heart and on the development of myocarditis.

Monoclonal antibodies to human immunoglobulin M (IgM) were used in a four-phase enzyme immunofluorescence "capture" assay for determination of IgM antibodies to measles and rubella viruses. Little or no background reactivity was seen in the test system, and interfering effects of rheumatoid factor were avoided by preabsorption of test sera with aggregated human IgG. Virus-specific IgM antibody was demonstrable in 23 of 24 patients with serological evidence of measles virus infections and in 36 of 36 patients with serological evidence of postnatal rubella infection. A few of the rubella patients did not show IgM antibody until 5 days after onset of illness. The enzyme immunofluorescence assay was able to demonstrate rubella IgM antibody in congenitally infected newborns, whereas indirect immunofluorescence results for virus-specific IgM were negative. Viral IgM antibody was not detected in persons with past infections with the test viruses, in young children without evidence of past infection, or in patients infected with heterotypic viruses, rickettsiae, chlamydiae, or mycoplasmas.

The presence of specific antibodies to human herpesviruses in certain purified human IgG preparations from commercial sources may produce misleading results when such preparations are used in studied on the IgG receptors produced by virus-infected cells.

Monoclonal antibodies conjugated with fluorescein permitted rapid, strain-specific identification of influenza A isolates and type-specific identification of influenza B isolates by direct immunofluorescence staining. Identification of H1 influenza A strains could be accomplished by direct immunofluorescence on cell culture fluids lacking sufficient hemagglutinin activity to permit identification by hemagglutination inhibition.

Infection of fibroblast cell lines initiated from BALB/c or NFR mice with coxsackievirus B3 (CBV-3) or B4 (CBV-4) resulted in infections which persisted for a limited number of subpassages of the infected cells in most cases, but for over a year in one case. In all instances primary acute infections were characterized by cytopathology and release of infectious virus progeny. Viral antigen could be detected during the acute phase of infection, but not in subcultured infected cells. Infectious center assays showed that every cell was infected during the acute phase of infection, but that from the first subcultivation on, the numbers of cells which were able to initiate infection were greatly reduced. The long term persistent CBV-3 infection was characterized by wide fluctuations in titers of virus released into the supernatant fluids. Interferon did not appear to play a role in maintenance of the persistent infection. Information derived from studies on mechanisms of CBV persistence in the in vitro model may help to elucidate the role of CBV in chronic human diseases such as myocarditis.

The RD cell line, derived from a human rhabdomyosarcoma, supported replication of a number of group A coxsackieviruses, including types A5 and A6 which heretofore have been propagable only in suckling mice. A number of the group A coxsackievirus types which replicated in RD cells had higher titers in this cell line than in other cell culture systems. In tests on a limited number of clinical specimens, RD cells were slightly less sensitive than suckling mice for isolation of group A coxsackieviruses, but they did permit the recovery of certain virus types which previously could be isolated only in suckling mice. Group B coxsackieviruses replicated poorly or not at all in RD cells.

Thirty-seven clinical isolates of coxsackievirus (CV) serotypes B-1, B-3, B-4, and B-5 were inoculated into male SJL mice. Twelve strains resulted in minor abnormalities of glucose metabolism in one or more of six infected mice (Tables 1 and 2). Sequential infection of male SJL mice with CVB-3, CVB-4, and CVB-5 resulted in abnormal glucose metabolism in 25 percent of the mice (Fig. 1). The glucose index of the abnormal animals was similar to that produced by sequential infection with reovirus and cytomegalovirus but less than that seen with more severe beta cell tropic agents such as streptozotocin or encephalomyocarditis virus. Infection of autoimmune New Zealand (NZB X NZW) F1 male mice with CBV-3, CVB-4, and CVB-5 resulted in transient elevation of the blood glucose concentration associated with acute acinar pancreatitis (Fig. 2). In spite of recent evidence that infection with the coxsackie B viruses can result in human diabetes mellitus, the diabetogenic potential of CVB field strains appears to be limited. Diabetes mellitus may occur as a rare event, limited to genetically susceptible hosts. Autoimmune mechanisms or repeated infection with other CVB serotypes may convert minimal beta-cell destruction into clinically overt disease.

An enzyme immunossay (EIA) was adapted for detection of antibody to varicella-zoster virus, and its sensitivity and specificity were compared with those of neutralization, immune adherence hemagglutination (IAHA), and complement fixation tests. Test sera showed little nonspecific reactivity in the EIA system, and valid results could usually be obtained at serum dilutions as low as 1:8. Demonstration of the presence or absence of varicella-zoster viral antibody by EIA showed 94% correlation with results obtained in neutralization tests, but EIA titers were 2- to 16-fold higher than neutralizing antibody titers. Results by IAHA showed 87% correlation with those obtained by neutralization. No false positive IAHA results were seen, but a number of false negative IAHA results were seen at the 1:8 serum dilution, particularly in older individuals. With increasing age (>40 years), and presumably increased time from varicella infection, neutralizing antibody levels generally declined to 1:8 or 1:16, EIA levels fell to 1:128 or 1:256, and IAHA and complement fixation antibody titers were usually <1:8 or 1:8. EIA and IAHA were as reliable as the neutralization and complement fixation tests for serodiagnosis of varicella and zoster infections. All tests demonstrated heterotypic varicella-zoster antibody titer rises in selected patients with initial herpes simplex virus infections, but fewer heterotypic responses were seen by EIA than by the other methods. EIA offers a rapid, sensitive, and specific method for varicella-zoster antibody assay that is applicable to use in a clinical setting.

The anticomplement immunofluorescence (ACIF) test was compared with the fluorescent antibody-to-membrane antigen (FAMA) test for determining varicella-zoster virus antibody levels as a measure of varicella-zoster virus immunity status. The ACIF test was found to be comparable to the FAMA test in sensitivity and could be used for examining sera at low dilutions of 1:2 and 1:4. In addition, the ACIF method proved to be a more economical procedure in terms of antigen required and personnel time necessary to perform the test. Heterologous varicella-zoster virus antibody titer rises were demonstrated by the FAMA test with 10 serum pairs from patients with clinically diagnosed genital herpes simplex virus infection, indicating that the FAMA test is no more suitable than other serological methods for serodifferentiation of those herpes simplex virus and varicella-zoster virus infections in which antibody increases occur to both antigens.

Studies were performed to define conditions under which propagation, assay and stabilization of the Delta herpesvirus (DHV) strain of simian varicella virus might be improved, and to compare biological properties of DHV with those of human varicella zoster virus (VZV). A mycoplasma contaminant was successfully eliminated from the DHV seed virus by treatment with a specific anti-serum. DHV was found to replicate more efficiently in the BS-C-1 line of African green monkey kidney cells than in Vero cells, and seed virus preparations in the form of virus-infected cells were produced which had infectivity titers greater than or equal to 1 X 10(6) p.f.u./ml. Greater yields of virus were produced in cultures infected as dispersed cells than as preformed monolayers. Infectious DHV could be released from host cells by sonic treatment of heavily infected cultures at 48 h post infection. Certain agents reported to enhance replication of herpes viruses (caffeine, carbaryl, the tumor promoter 12-0-tetra-decanoyl-phorbol-13-acetate, and DEAE-dextran) had no enhancing effect on replication of DHV. However, DEAE-dextran in the maintenance medium enhanced spontaneous release of DHV into culture fluids. Plaquing efficiency and plaque size of DHV were greater in BS-C-1 than in Vero cells, and plaque assays and plaque reduction neutralization tests were developed in this cell system using a solid overlay medium with neutral red vital stain. Neutralization of DHV was markedly enhanced by fresh guinea pig complement. The newly developed neutralization test demonstrated more vigorous antibody responses to DHV in active and latent VZV infections than were demonstrated with previous procedures. In addition to their preferential growth in monkey and human cells respectively, DHV and VZV were found to differ markedly in their rates of attachment to host cells, with DHV requiring over 6 h of adsorption, while VZV adsorption was essentially complete at 1 h. Also, cell-free DHV was much more resistant than cell-free VZV to repeated cycles of freezing and thawing.

In 1975 in Bulgaria a severe epidemic of central nervous system (CNS) disease occurred. Clinically, histopathologically, and epidemiologically the cases resembled poliomyelitis, aseptic meningitis, meningoencephalitis, and, in some cases, encephalomyocarditis. About 21% of the 700 reported cases developed paralysis, 44 with fatal outcome [ref. 1]. In 65 cases, 92 strains of enterovirus of the same serologic type were isolated: 38 strains from the CNS, 10 from mesenteric lymph nodes and tonsils, and 44 from feces [ref. 1,2]. A typical representative strain, No. 258, isolated from the spinal cord of a 3-month-old baby who died on the 5th day of disease with signs of focal polioencephalitis, was selected for intensive study. Cross-neutralization tests established the antigenic identity of the Bulgarian 258 strain (Stanchev) with American strains of enterovirus type 71 (BrCr and JaFr strains) and Swedish strains of the same type (Nos. 52343, 52500, and 6041). From its biological [ref. 1,2], physicochemical [ref. 3], and antigenic properties, the etiological agent of this large epidemic can be classified as a member of enterovirus type 71, one of the most highly pathogenic of the recently recognized enteroviruses.

Foci of cells infected with human cytomegalovirus were noted to stain more intensely than uninfected cells with neutral red, and this provided the basis for development of a plaque assay and plaque reduction neutralization test for cytomegalovirus. Plaques demonstrable by neutral red staining could be counted at 8 days after infection; thus, results could be obtained earlier than for plaque assay systems based upon the viral cytopathic effect, a fewer manipulations were required for staining cell monolayers to demonstrate plaques. Certain variables affecting plaque size and numbers and antibody titers were defined. Addition of fresh guinea pig complement to the reaction mixtures markedly enhanced cytomegalovirus-neutralizing antibody titers of hyperimmune animal sera, but titers of human sera were enhanced only two-or fourfold.

A murine model system for evaluation of myocarditic and cardiotropic properties of strains of group B, type 4 coxsackievirus (CBV-4) was developed in male BALB/c mice 4 weeks of age. Differing cardiotropic and myocarditic properties could be identified among field strains within the CBV-4 serotype. These properties were consistent for the virus strains, and were independent of the infecting virus dose. Virus replication in the heart appeared to be essential for development of myocarditis, but some infected hearts with high levels of infectious virus did not show myocarditis. Two of the myocarditic strains showed different histopathology in infected hearts; with one strain (Mil) the myocarditis was characterized by a marked inflammatory reaction with occasional accompanying myofiber necrosis. With the other strain (Bol), necrosis was the predominant finding, with a much lesser degree of inflammation. These findings suggest that there may be various pathogenic or immunopathogenic mechanisms by which CBV-4 can produce myocarditis.

Monoclonal antibodies to human IgM were produced by fusing the Sp 2/0-Ag 14 line of mouse myeloma cells with spleen cells from BALB/c mice immunized with purified human IgM. From 6 clones which secreted antibody to human IgM, the one which produced the highest levels of antibody and grew relatively rapidly was selected for expansion and production of immune reagents for viral IgM antibody assays. Mouse ascitic fluids produced with this clone of cells had antibody titers for human IgM of 1 X 10(-10) by indirect enzyme immunofluorescence assay (EIFA). The monoclonal antibodies were found to belong to subclass 1 of murine IgG, and their specificity was shown to be directed against the Fab portion of the mu chain of human IgM. Antibodies from murine ascitic fluid conjugated with horseradish peroxidase were shown to be suitable for assay of measles IgM antibody by indirect immunoperoxidase staining. Antibodies conjugated with alkaline phosphatase could be used in an indirect EIFA for determination of measles IgM antibodies; use of monoclonal conjugates in this system eliminated the nonspecific activity observed in tests utilizing polyclonal anti-mu reagents. Further, the monoclonal antibodies were highly satisfactory for use in a 'capture' system for viral IgM antibody assays. The availability of monoclonal antibodies to human IgM overcomes problems with specificity, consistency and supply which have previously hindered development and standardization of viral IgM antibody assays.

Envelope, capsid, and soluble antigens of Herpesvirus hominis (HVH) types 1 and 2 were compared to crude antigens (disrupted HVH-infected cells) for potency, type-specificity, and diagnostic value in the indirect hemagglutination (IHA) test. The envelope appeared to be the predominant component reacting in the IHA test, but recurrent HVH infections increased the reactivity of human sera with capsid antigens. The soluble antigens reacted in the IHA test with HVH immune animal sera, but very few convalescent-phase human sera showed reactivity with soluble antigens. Overall, none of the subunit antigens showed greater type-specificity than did crude antigens in IHA tests with immune animal or convalescent-phase human sera. Recurrent infections with type 1 or type 2 viruses tended to broaden heterotypic reactivity of the patients' sera with both crude and subunit antigens, even in patients showing only a single type of antibody by IHA inhibition. Subunit antigens were no more sensitive than crude antigens in demonstrating significant IHA antibody titer rises for serodiagnosis of herpesvirus infections, and they generally had to be used at lower working dilutions than crude antigens.

Enzyme immunofluorescence assays for cytomegalovirus antibodies could be read satisfactorily using a light box with ultraviolet illumination. Higher antibody titers were obtained with a fluorogenic substrate than with a color-producing substrate.

Antibody titers to cytomegalovirus were determined in 204 single-serum specimens and in 138 serum pairs, utilizing as antigen extracts of infected cells prepared by either freezing and thawing cells (FT antigen) or extracting them with a glycine buffer (GE antigen). With the serum pairs, 40% of the early sera and 44% of the late sera showed a fourfold or higher antibody titer with the GE antigen compared with those with the FT antigen, whereas 39% of the single sera had a fourfold or higher titer with the GE antigen. Sucrose density gradient centrifugation of the two antigen preparations indicated that the GE antigen contained two peaks of complement-fixing activity, one at a density of 1.18 to 1.22 g/cm3 and one at 1.06 to 1.10 g/cm3. The FT antigen had only one discernible peak of complement-fixing activity at 1.04 to 1.10 g/cm3. On electron microscopy, the more dense peak contained mainly nucleocapsids with some dense bodies and occasional enveloped virions, and the lighter peak was composed of amorphous material. Antibody titers obtained with the crude GE antigen and the more dense peak (designated viral antigen) were in agreement, whereas titers with the FT antigen agreed with those obtained with the lighter peak (soluble antigen). The antibody detected with both antigens was mainly immunoglobulin G.

To explore the role of viruses in the etiology of diarrhea in colony-reared monkeys, direct electron microscopy, the fluorescent virus precipitin test and cell culture inoculation were used to examine the stools of monkeys with and without diarrhea. The animals were predominantly rhesus with a few macaques of other species, and included infants, juveniles and adults. Adenoviruses were isolated from a higher proportion of specimens from rhesus monkeys with diarrhea (73% of specimens from infants and 78% of specimens from juveniles and adults) than from control monkeys without diarrhea (22% of specimens from infants and 26% of specimens from juveniles and adults). SV 20 was the most frequently isolated simian adenovirus type; SV 17 and SV 32 also were recovered. Noncultivable adenoviruses detectable only by electron microscopy were not seen. Although adenovirus excretion was associated with diarrhea, the causal role of adenoviruses was difficult to assess. When serial specimens from animals with chronic or intermittent episodes of diarrhea were examined, sequential infections with different viruses were found to be common. Rotaviruses were detected by electron microscopy and isolated in cell cultures from two infant rhesus monkeys with diarrhea. However, the low detection rate, together with negative serologic data on 40% of infant monkeys with diarrhea, suggested that rotaviruses were not the major cause of gastroenteritis in the monkeys under study.

The indirect immunofluorescence test was used to measure immunoglobulin M (IgM) and IgG antibodies to acetone-fixed Ureaplasma urealyticum organisms in sera from 128 adults with genital infections and from 713 symptomatic newborns and babies 1 day to 18 months old. Thirty-four percent of the adults had demonstrable IgG antibody to ureaplasma. IgM antibody was detected in 2 of the adult sera and in 17 of the infant sera. These babies were divided into two distinct groups. Ten of the infants presented at birth with various physical findings, whereas the onset of symptoms for the other 7 occurred 3 to 13 weeks after birth, and the major clinical finding in 6 of the 7 was respiratory distress. The results of this study suggested that U. urealyticum infection may be associated with fetal damage and infant pneumonia, and if this is substantiated, the indirect immunofluorescence test employing acetone-fixed antigen to measure IgM antibody to U. urealyticum may be an important diagnostic tool.

To explore possible advantages which immunoperoxidase (IP) staining might have over immunofluorescence (IF) staining for identifying rubella virus isolates, direct comparative studies were done on the same coded clinical materials using the same rubella immune rabbit serum as the primary antiserum in both systems. The rubella immune rabbit serum and conjugated anti-rabbit immune globulins could be used more dilute in the IP system than in the IF system. Both IP and IF staining detected rubella antigen in all specimens which were positive by interference. IP staining also detected low levels of rubella antigen in a few additional specimens which had originally been positive for rubella virus, but which on retesting were negative by interference and IF staining. With second-cell-culture-passage material, IP and IF staining showed comparable specificity, and the few specimens which reacted nonspecifically generally did so in both systems. Cell cultures inoculated directly with urine specimens showed greater nonspecificity by IP than by IF, but this activity could be abolished by pretreatment with sodium azide and peroxide; other methods tried for inactivating endogenous peroxidase activity destroyed rubella antigen as well. The intensity of staining for positive specimens was comparable in the two systems. However, more antigen was demonstrable in both systems when BHK-21 cells were inoculated as a cell suspension and then permitted to grow into monolayers than when the same specimens were inoculated into preformed monolayers. IP staining was considered to be a highly satisfactory alternative to IF staining for identification of rubella virus isolates.

NFR nude (nu/nu) and euthymic (+/nu) littermates were infected with coxsackievirus B3 (CBV-3) and assayed for virus persistence in the heart, pancreas and spleen, for development of myocarditis and for antibody production. The virus grew to higher titer and persisted longer in hearts of nu/nu mice. In both types of mice there was comparable myocarditis with a mononuclear cell inflammatory response, and there was some evidence of chronic lesions for up to 21 days in +/nu and 28 days in nu/nu mice. Antibody of the IgM class was present at 7 days in both strains of mice. Thereafter, +/nu mice produced high titers of virus-specific IgG antibody, while nu/nu mice produced little or no viral IgG antibody. The persistence of virus for up to 28 days in NFR nude mice is longer than has been reported previously, and offers an opportunity for further study of the role of T-cells in virus persistence and myocarditis.

There has been much interest and activity in the development of techniques for rapid viral diagnosis which would allow successful intervention in the treatment of patients or their contacts or in the control of viral diseases in the community. The greatest emphasis has been on techniques that permit viral detection directly in the clinical specimen, since these avoid the need to cultivate the agent, are feasible for detection of viruses that cannot be cultivated, and can detect virus in specimens in which the agent is no longer infectious. Direct methods used for viral detection include electron microscopy and various immunoassays which are based on demonstrating reactivity of viral antigen in the specimen with known viral antisera. The use of immunoassays for more rapid identification of viruses isolated in laboratory host systems and for selective detection of viral antigen in inoculated cell cultures even before the agents produce an observable effect has been an important advance in viral diagnosis by the approach of isolation and identification. The reliability of all specific viral identification procedures depends on the use of high quality viral antisera. Some of the problems previously encountered in preparing satisfactory viral immune reagents are being overcome through the availability of highly specific monoclonal antibodies produced by cell hybridization techniques. Virus-specific IgM antibody assays for rapid diagnosis have been improved greatly through the use of a "capture" technique in which antibody to the human mu chain is used in the solid phase to separate IgM from other serum components which might compete with IgM antibody or give nonspecific reactivity, and also through the availability of highly specific monoclonal antibodies to the human mu chain. A variety of simple assays for determination of viral antibody status have been developed, and many are commercially available. The reliability of some of these antibody assays has been improved through the incorporation of more suitable controls and through better definition of interpretations which should be made from test results.

Indirect hemagglutinating (IHA) antibody responses to Herpesvirus hominis types 1 and 2 (HVH-1 and HVH-2) were compared to complement-fixing and neutralizing antibody responses in immunized laboratory animals (rabbits, guinea pigs, and hamsters) and in natural infections of man. With the immunized animals, type specificity was seen only in the IHA test and only with antisera produced in hamsters and in the rabbits immunized with HVH-2. In human nongenital infections (considered to be caused predominately by HVH-1), IHA and neutralizing antibodies developed at about the same rate and reached approximately the same levels for HVH-1 and HVH-2. IHA titers tended to be higher than neutralizing antibody titers for both virus types. In genital infections (considered to be caused predominately by HVH-2), there was a rapid IHA antibody response to HVH-2, and the early HVH-2 antibody demonstrable by IHA, but not by neutralization tests, was found to be immunoglobulin M in nature. In genital infections, IHA titers for HVH-2 were markedly higher than neutralization titers, but there was no pronounced difference in neutralizing the IHA antibody titers for HVH-1. Several patients with genital infections fialed to develop IHA antibody for HVH-1. The IHA test possessed no greater sensitivity than did complement fixation or neutralization tests for serodiagnosis of HVH infections. Despite the fact that a number of patients with genital infections produced IHA antibody only for HVH-2, the test was no more effective than the neutralization test in providing a type-specific serodiagnosis of infection, due largely to the fact that the rapid IHA antibody response to HVH-2 prevented demonstration of a further, significant antibody titer increase in a number of cases.

The Delta herpesvirus (DHV) which produced outbreaks of exanthematous disease in patas monkeys was shown to have a close immunological relationship to varicella-zoster (V-Z) virus of man. Immunization of rhesus monkeys with DHV or V-Z virus resulted in the development of neutralizing antibodies to both viruses and also in the production of complement-fixing antibodies to V-Z virus. Immunoglobulin M neutralizing antibody to V-Z virus was demonstrated in the serum of a rhesus monkey immunized with DHV, suggesting a primary antibody response rather than recall of antibody to a related virus. Convalescent-phase sera from human zoster cases had comparable levels of neutralizing antibody for both DHV and V-Z virus. Patas monkeys involved in an outbreak of DHV infection showed seroconversion to both DHV and V-Z virus by neutralization tests and to V-Z virus by complement fixation tests. The demonstration of the close antigneic relationship between DHV and V-Z virus suggests that DHV may be useful in an animal model system for studies on the latency and reactivation of V-Z virus.

Pretreatment of host cells with carbaryl delayed the early spread of simian and human varicella virus infections. Toward the end of the growth cycle there was an apparent enhancement of infection, since treated cultures showed more infectious centers than did untreated ones in which infectivity had reached maximum levels and then declined.

Various subunit antigens of varicella-zoster (V-Z) virus were examined for complement-fixing (CF) activity with sera from homotypic infections and from herpes simplex virus (HSV) infections in which a CF antibody titer rise was demonstrated with crude V-Z antigen. The subunit antigens included nucleocapsids, envelopes, a soluble antigen produced from infected culture fluids by sucrose density gradient centrifugation, a soluble antigen produced by reducing the volume of clarified infected culture fluids, a soluble antigen derived from infected cell lysates, a "viral" antigen consisting largely of enveloped particles with a few nucleocapsids, and a cell membrane-associated antigen. None was more suitable than crude V-Z antigen for serodifferentiation of V-Z virus and HSV infections. The envelope antigen, cell membrane antigen, and the soluble antigen prepared by density gradient centrifugation showed little reactivity with sera from varicella and HSV infections, but gave high antibody titers with sera from zoster infections, suggesting that a secondary V-Z virus infection is required to produce an antibody response to these subunit antigens. Patients with varicella and zoster infections and the selected patients with HSV infections all showed significant CF antibody responses to the other V-Z subunit antigens.

Systematic studies on the replication of varicella-zoster virus in infected human fetal diploid lung cells have defined more optimal conditions for infection and harvesting of cultures and have led to the production of cell-free virus preparations with infectivity titers of greater than or equal to 10(6) plaque-forming units per ml. The highest yields of cell-free virus were obtained by (i) sonic treatment of the cellular phase of cultures inoculated with trypsin-dispersed infected cells at ratios of 1 infected cell to 6 to 10 uninfected cells in the monolayer and (ii) harvesting cells after 24 to 36 h of incubation at 36 degrees C. At this time the cultures showed minimal viral cytopathic effect. Spread of infectivity occurred much more rapidly in cultures inoculated with whole infected cells than in those infected with cell-free virus. Complement-fixing antigens with improved titers of greater than or equal to 1:128 were prepared from varicella-zoster virus-infected cell cultures in the same manner as cell-free virus, but harvested after 3 to 4 days of incubation when the cultures showed an advanced cytopathic effect.

Monoclonal antibodies to varicella-zoster virus were used to study viral glycoproteins by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on the viral glycoproteins immunoprecipitated, the five monoclonal antibodies fell into three groups. Two antibodies, 4B7 and 8G9 (group 1), immunoprecipitated a single glycoprotein of molecular weight (MW) 118,000 (118K glycoprotein) and had high neutralizing activity in the absence of complement. One antibody, 3C7 (group 2), which lacked neutralizing activity, immunoprecipitated two glycoproteins of MWs 120,000 and 118,000 and a glycoprotein giving a diffuse band in the region of 64,000 to 65,000. Pulse-chase experiments and experiments with monensin as an inhibitor of glycosylation suggested that the 120K polypeptide was derived by glycosylation of the 118K polypeptide and that a 43K antigen was processed into the 64 to 65K glycoprotein. Two antibodies, 3G8 and 4E6 (group 3), both had neutralizing activity only in the presence of complement, and both immunoprecipitated at least five polypeptides, with MWs ranging from 50,000 to 90,000. Antibody 3G8 was isotype immunoglobulin G2b (IgG2b), and its immunoprecipitating activity was stronger than that of 4E6, which was isotype IgG1. Pulse-chase experiments with antibody 3G8 showed that lower-MW glycopeptides chased into three polypeptides of MWs 90,000, 80,000, and 60,000 by 24 h. Immunoprecipitation experiments with antibody 3G8 on infected cells treated with glycosylation inhibitors 2-deoxyglucose, monensin, and tunicamycin, suggested that a prominent, early-appearing 70K polypeptide may have been processed into the glycoproteins of higher MWs and that the 60K polypeptide may have been derived by glycosylation of polypeptides of lower MWs.

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.

Neutralization of varicella-zoster (V-Z) virus by human sera and immune rhesus monkey sera was enhanced by fresh guinea pig complement. There was no marked difference in the degree to which complement enhanced neutralization by sera from current V-Z virus infections and sera from long-past varicella infections. Immunoglobulin G neutralizing antibody in sera from varicella cases was enhanced by complement to a slightly higher degree than was immunoglobulin M (IgM) antibody, and immunoglobulin G neutralizing antibody in immune monkey sera was enhanced to a much greater degree than was IgM antibody. There was a rapid decline in the complement requirement of IgM neutralizing antibodies over the course of immunization of the rhesus monkeys. V-Z neutralizing antibody titers in the presence of complement were higher than complement-fixing titers of the same sera in all groups of individuals studied. IgM neutralizing antibody for V-Z virus was demonstrable in all cases of varicella but in only 1 of 22 zoster cases, and V-Z IgM neutralizing antibody was not detectable in primary herpes simplex virus infections in which heterotypic antibody titer rises occurred to V-Z virus. Complement-fixing antibody for V-Z virus was absent in 19S serum fractions which contained IgM neutralizing antibody for the virus.

We have studied the epidemiologic characteristics of insulin-dependent (Type 1) diabetic patients aged 0-19 in a city (San Diego, southern California, USA) characterized by an impressive racial diversity and especially mild and constant climatic conditions. Ascertainment was through retrospective review of medical records in 19 hospitals. For the 3 years 1978-1981 the mean annual incidence of diabetes was 7.3 cases/100,000, with no statistical difference between the sexes. The observed incidence rates in the various ethnic groups was significantly different from expected (p less than 0.03), with an excess of cases among Caucasians and fewer than expected cases among Mexicans, Blacks and Orientals. There was no identifiable seasonal trend. Some of the clinical characteristics at diagnosis differed between the sexes: males were slightly older (9.3 +/- 5.2 years versus 8.8 +/- 3.9 for females), had a shorter duration of diabetes-related symptoms and a higher frequency of infections both at the time of diabetes diagnosis and in preceding months. Females tended to have a higher frequency of Type 1 diabetes in first-degree relatives. This study documents for the first time that, among multiple racial groups living in the same environment, Caucasians are at the highest risk of developing juvenile-onset Type 1 diabetes.

Cross-neutralization and complement fixation tests demonstrated the immunological identity of the Delta herpesvirus, the 592S virus, the Liverpool vervet monkey virus, the herpesvirus of patas monkeys, and the Medical Lake macaque virus. These viruses were isolated from diverse outbreaks of varicella-like disease in simians and from various simian species. All of the simian viruses were shown to be related to human varicella-zoster (V-Z) virus, as evidenced by the fact that immunization of monkeys with each of the simian viruses elicited the production of both neutralizing and complement-fixing antibodies to V-Z virus. However, cross-complement fixation tests indicated that the simian viruses are not so closely related to V-Z virus as they are to one another. Varicella or zoster infections in humans produced neutralizing and complement-fixing antibody responses to each of the simian viruses; the responses were more marked in zoster infections than in varicella infections but, in most patients, antibody levels produced to the simian viruses were much lower than those to the homologous V-Z virus.

Results by an enzyme immunoassay method (EIA) performed at one serum dilution and results by indirect immunofluorescence (IFA) and hemagglutination inhibition (HI) tests performed at step dilutions were correlated with results by a neutralization test (50% plaque neutralization [PN]) performed at step dilutions on single serum samples for serologic evaluation of immunity status to measles virus. PN results were taken as true indicators of immunity, and the other tests were evaluated on that basis. The predictive value of a positive result being positive also by PN was 95.3% for HI and 93.3% for EIA and IFA. The predictive value of a negative result being negative also by PN was 81.1% for HI, 100% for EIA, and 75.0% for IFA. A similar study on immunity status to varicella-zoster virus by EIA and by an anticomplement immunofluorescence test versus PN showed a 100% predictive value of a positive or negative result by EIA. By the anticomplement immunofluorescence test, the predictive value of a positive result was 97.7%, and that of a negative result was 88.5%. Studies on the comparative ability of EIA versus complement fixation (CF) to detect significant changes in antibody concentration between acute-phase and convalescent-phase serum samples indicative of a current infection were also done. Both tests were satisfactory for the serodiagnosis of measles or varicella-zoster virus infections. However, EIA was preferable to CF because it was less technically difficult, less labor intensive, and could be performed on sera that were anticomplementary in CF reactions.

Direct immunofluorescence (IF) staining was compared with virus isolation for detection of herpes simplex viruses (HSV) and varicella-zoster virus (VZV) directly in clinical materials. These included 199 vesicular lesion specimens and 280 tissue specimens. Correspondence between IF and isolation results was 88% in testing for HSV in lesion specimens and 98% in testing for HSV in various tissue (mostly brain) specimens. Overall, IF was positive for 82% of the specimens in which HSV was demonstrated, and virus was isolated from 89% of the HSV-positive specimens. IF was markedly more sensitive than isolation for demonstrating VZV in lesion and tissue specimens, detecting all of the specimens positive for VZV, whereas isolation detected only 23%. IF detected VZV antigen in a number of lesion specimens taken late after onset, past the time when they would be expected to yield infectious virus. Specificity of positive IF reactions for HSV or VZV in the absence of virus isolation was supported by the facts that (i) staining was obtained with only a single, presumably homologous, immune conjugate, (ii) clinical symptoms were compatible with infection with the virus for which positive IF findings were obtained, and (iii) positive electron microscopy findings for herpesviruses or positive serological results for VZV were also obtained in some instances. Factors to be considered in achieving specificity of IF staining for these human herpesviruses are discussed.