Susceptibility of bovine macrophages to infectious bovine rhinotracheitis virus infection.

Infectious bovine rhinotracheitis virus replicated in cultured bovine alveolar macrophages (AM). However, yields of infectious virus were low, with maximum titers approximately 100 times that of the residual inoculum. Immunofluorescence and electron microscopic studies indicated that the majority of macrophages produced viral antigen, but after infection at a multiplicity of 0.1, only 4.1% of AM produced infectious centers. Virus-infected AM culture supernatants possessed interfering activity, probably due to interferon. Incubation of fresh AM with these fluids rendered them refractory to infection. Although AM from infectious bovine rhinotracheitis virus-immune and -susceptible donors were equally permissive and their susceptibility was unaltered by incubation with bacterial lipopolysaccharide, bovine mammary macrophages which were elicited with lipopolysaccharide became nonpermissive when further incubated for 48 h with 1 microgram of lipopolysaccharide per ml. Under these conditions, infected mammary macrophages failed to synthesize viral DNA, and there was reduced synthesis of "late" viral polypeptides.     

Antipneumococcal effects of C-reactive protein and monoclonal antibodies to pneumococcal cell wall and capsular antigens.

Antibodies to pneumococcal capsular polysaccharides are well known for their ability to protect against pneumococcal infection. Recent studies indicate that antibodies to cell wall antigens, including pneumococcal surface protein A and the phosphocholine (PC) determinant of teichoic acids as well as human C-reactive protein (which also binds to PC), can protect mice against pneumococcal infection. In the present study we compared the protective effects of these agents as measured by mouse protection, the blood bactericidal assay, and clearance of pneumococci from the blood and peritoneal cavity. Our findings extend previous results indicating that human C-reactive protein and antibodies to noncapsular antigens are generally less protective than anticapsular antibodies. The new results obtained indicate the following: (i) mouse protection studies with intraperitoneal and intravenous infections provide very similar results; (ii) monoclonal immunoglobulin G2a (IgG2a) antibodies to PC, like IgG1, IgG2b, and IgG3 antibodies to PC, are highly protective against pneumococcal infection in mice; (iii) human antibody to PC is able to protect against pneumococcal infection in mice; (iv) antibodies to PspA are effective at mediating blood and peritoneal clearance of pneumococci; (v) complement is required for the in vivo protective effects of both IgG and IgM antibodies to PC; (vi) IgG1, IgG2b, and IgG3 anti-PC antibodies all mediate complement-dependent lysis of PC-conjugated erythrocytes; and (vii) antibodies and human C-reactive proteins that are reactive with capsular antigens but not cell wall antigens are able to mediate significant antibacterial activity in the blood bactericidal assay.     

The vector homology problem in diagnostic nucleic acid hybridization of clinical specimens.

Nucleic acid hybridization techniques using cloned probes are finding application in assays of clinical specimens in research and diagnostic laboratories. The probes that we and others have used are recombinant plasmids composed of viral inserts and bacterial plasmid vectors such as pBR322. We suspected that there was material homologous to pBR322 present in many clinical samples. because hybridization occurred in samples which lacked evidence of virus by other techniques. If the presence of this vector-homologous material was unrecognized, hybridization in the test sample might erroneously be interpreted as indicating the presence of viral sequences. In this paper we demonstrate specific hybridization of labeled pBR322 DNA with DNA from various clinical samples. Evidence is presented that nonspecific probe trapping could not account for this phenomenon. In mixing experiments, it is shown that contamination of clinical samples with bacteria would explain such a result. Approaches tested to circumvent this problem included the use of isolated insert probes, alternate cloning vectors, and cold competitor pBR322 DNA in prehybridization and hybridization mixes. None proved entirely satisfactory. We therefore emphasize that it is essential that all hybridization detection systems use a control probe of the vector alone in order to demonstrate the absence of material with vector homology in the specimen tested.     

A comparison of fluorescein isothiocyanate and lissamine rhodamine (RB 200) as labels for antibody in the fluorescent antibody technique

The relative merits of fluorescein isothiocyanate (FITC) and lissamine rhodamine (RB 200) as labels for antibody in fluorescence microscopy were studied and compared by microphotometry, testing each fluorochrome under its own optimal conditions as far as possible, and at a similar range of dye:protein ratios. The antibody was sheep anti-human globulin, and the tissues stained with it were rat liver sections bearing human anti-nuclear factor on the nuclei. The findings were as follows:

(i) the amount of RB 200 conjugating with protein was strictly proportional to the amount of the sulphonyl chloride derivative added to the reaction mixture; with increasing amounts of FITC in the reaction mixture, however, there was a less than proportional increase in the degree of conjugation.

(ii) Diethylaminoethyl (DEAE)-cellulose chromatography decreased the dye:protein ratio of the conjugates by 40% uniformly for both RB 200 and FITC, regardless of the initial dye:protein ratio.

(iii) When corrections were made for spectral responses of photo-detectors, effects of optimizing the mountants, and benefits to rhodamine of changing from a Xenon to a mercury lamp, it was concluded that RB 200 conjugates could give brighter staining than FITC conjugates at similar dye:protein ratios.

(iv) DEAE-cellulose chromatography greatly improved the contrast of the staining, especially with RB 200 conjugates.

(v) After chromatography, RB 200 consistently gave better contrast than FITC.

(vi) The fluorescence of rhodamine-stained sections did not fade demonstrably when irradiated for several minutes with green light.

(vii) The fluorescence of FITC-stained sections faded rapidly when irradiated with ultra-violet (u.v.)+blue light. The fluorescence appeared to contain two components, one fading with first-order kinetics with a half-life of about a minute under the experimental conditions used and the other not fading at all.

(viii) Raising the pH improved the fluorescence of FITC-stained sections but did not affect rates of fading.

(ix) Narrow-band excitation of FITC-stained sections with blue light instead of u.v.+blue reduced the rate of fading and the fluorescence intensity by equal amounts, an effect presumably due merely to loss of excitation intensity.

     

Culture confirmation of cytomegalovirus and herpes simplex virus by direct enzyme-labeled DNA probes and in situ hybridization.

Using probes consisting of horseradish peroxidase (HRP) directly attached to DNA, scrapings or trypsinized cells from 217 adequate clinical samples were cultured and analyzed in 3 blind studies by in situ hybridization for the presence of cytomegalovirus (CMV) and herpes simplex virus (HSV). Sixty samples were judged inadequate due to insufficient cell numbers; however, this problem was significantly decreased during the course of the study. One hundred and eighteen samples were found positive and 70 samples were found negative for CMV. Scrapings of cultured cells from 29 clinical samples revealed 9 samples which were positive and 20 samples which were negative for HSV. Forty-two additional samples, containing either uninfected cells or cells infected with various strains of CMV, were analyzed for the ability of the HRP-DNA CMV probe to detect such isolates. Twenty samples were positive and 22 negative for CMV. No false-negatives or false-positives were observed for either CMV or HSV. In addition to the specificity noted above neither the CMV nor the HSV DNA probe hybridized to potential contaminants found in clinical specimens.     

Patterns of needle acquisition and sociobehavioral correlates of needle exchange program attendance in Baltimore, Maryland, U.S.A

OBJECTIVES: This study examined factors associated with obtaining syringes from a needle exchange program (NEP) and other safer sources in Baltimore, Maryland, U.S.A. DESIGN AND METHODS: A cross-sectional face-to-face survey was administered to 741 current drug injectors recruited by snowball sampling techniques. A brief open-ended interview was conducted on a subsample. RESULTS: Most (85%) participants obtained needles from street needle sellers. Only 8% obtained their needles exclusively from safer sources (NEPs, pharmacies, hospitals, or patients with diabetes). Cocaine use was associated with obtaining needles from the NEP but not from exclusively safer sources. Obtaining needles from only safer sources was associated with being female and less frequent needle sharing and shooting gallery attendance. Among HIV-seropositive participants, those who were diagnosed before the year that the NEP began were more likely to obtain needles from safer sources. Participants who sold needles reported that it was easy to make used needles appear to be unused, and some admitted to selling used syringes as new. CONCLUSIONS: Street needle sellers are an important source of needles for drug injectors, and few injectors appear able to determine whether these needles are clean. Individual sealing of diabetic syringes may reduce the risk of blood-borne infections by enabling both drug injectors and patients with diabetes to better judge the sterility of the needles they purchase.     

How informed is consent? Use of an information booklet in patients undergoing total hip replacement

For informed consent, patients must understand the risks and benefits of the proposed procedure. Fifty patients undergoing total hip replacement (THR) participated in an evaluation of a newly devised information booklet. Patients' understanding of the risks and benefits of THR was assessed using a questionnaire before and after they had read the booklet. Pre-booklet patients knew on average 2 benefits of THR, compared with 3 afterwards. Twenty-two patients initially identified no alternative treatment to THR. Those who knew an alternative stated on average 1 treatment compared with 2 in the post-booklet group. Fifteen patients identified no risks of THR initially, compared with two after reading the booklet. Initially patients reported 1 risk, versus 3 after reading the booklet. Forty-eight patients thought that the booklet increased their understanding of THR and its risks. Fourteen found it upsetting, but acceptable. Forty-six patients wished to keep the booklet.     

Oral contraceptives and hepatocellular carcinoma

A series of 26 white women aged under 50 who developed hepatocellular carcinoma in a non-cirrhotic liver were studied for the possible role of oral contraceptives. Eighteen of the women had used the "pill" for a median of eight years. Over 1300 women whose use of the pill had been determined in another study served as controls. Patients and controls were divided into five age and four calendar groups and the relative risks associated with oral contraceptives calculated by multivariate analysis. Short term use of the pill was not associated with an increased risk of tumour development; nevertheless, use for eight years or more was associated with a 4.4-fold increased risk (p less than 0.01). When patients with markers of hepatitis B virus infection were excluded the relative risk was 7.2 (p less than 0.01). In both instances the absolute risk for developing hepatoma remained low.