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.

     

Allergic conversion of protective mucosal immunity against nasal bacteria in patients with chronic rhinosinusitis with nasal polyposis

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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.     

Human adipose-derived adult stem cells produce osteoid in vivo

Adult subcutaneous fat tissue is an abundant source of multipotent cells. Previous studies from our laboratory have shown that, in vitro, adipose-derived adult stem (ADAS) cells express bone marker proteins including alkaline phosphatase, type I collagen, osteopontin, and osteocalcin and produce a mineralized matrix as shown by alizarin red staining. In the current study, the ADAS cell ability to form osteoid in vivo was determined. ADAS cells were isolated from liposuction waste of three individual donors and expanded in vitro before implantation. Equal numbers of cells (3 x 10(6)) were loaded onto either hydroxyapatite/tricalcium phosphate (HA-TCP) cubes or the collagen/HA-TCP composite matrix, Collagraft, and then implanted subcutaneously into SCID mice. After 6 weeks, implants were removed, fixed, and demineralized and sectioned for hematoxylin and eosin staining. Osteoid formation was observed in 80% of HA-TCP implants loaded with ADAS cells. Only 20% of Collagraft implants were positive for the presence of osteoid matrix. Whereas 100% of HA-TCP implants loaded with hFOB 1.19 cells formed osteoid, Collagraft loaded with hFOB 1.19 cells displayed a high degree of adipose tissue within the matrix. Immunostaining of serial sections for human nuclear antigen demonstrated that the osteoid contained human cells. Osteoid formation was not observed in control HA-TCP or Collagraft matrices implanted without cells. In summary, the data demonstrate the ability of ADAS cells to form osteoid matrix in vivo. Because of their abundance and accessibility, ADAS cells may prove to be a novel cell therapeutic for bone repair and regeneration.     

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.     

A rational approach to the diagnosis of hypertrophic pyloric stenosis: do the results match the claims?

The currently accepted premise that the diagnosis of hypertrophic pyloric stenosis (HPS) should be made on clinical grounds, with ultrasound (US) and upper gastrointestinal series (UGIS) reserved for those with a negative clinical examination, was tested. Variable clinical skills of initial examiners, including pediatric surgeons, made abdominal palpation no more sensitive or specific than US or UGIS. For those with a negative clinical examination, proceeding directly to a UGIS will result in monetary savings, especially if good clinical performance decreases the probability of HPS among those without palpable pyloric "tumors." The benefits of a "US first" approach (no radiation, better patient and parent acceptance, no contrast medium) are less apparent but no less important and increase as clinical experience declines and performance of US improves. Criteria for the clinical or sonographic diagnosis of HPS should be kept strict to avoid false-positive results; false-negatives and other causes of vomiting should be identified by UGIS.     

Heterogeneous cardiac sympathetic denervation and decreased myocardial nerve growth factor in streptozotocin-induced diabetic rats: implications for cardiac sympathetic dysinnervation complicating diabetes

Heterogeneous myocardial sympathetic denervation complicating diabetes has been invoked as a factor contributing to sudden unexplained cardiac death. In subjects with diabetic autonomic neuropathy (DAN), distal left ventricular (LV) denervation contrasts with preservation of islands of proximal innervation, which exhibit impaired vascular responsiveness. The aims of this study were to determine whether this heterogeneous pattern of myocardial sympathetic denervation occurs in a rat model of diabetes and to explore a potential association with regional fluctuations in myocardial nerve growth factor (NGF) protein. Myocardial sympathetic denervation was characterized scintigraphically using the sympathetic neurotransmitter analog C-11 hydroxyephedrine ([11C]HED) and compared with regional changes in myocardial NGF protein abundance and norepinephrine content after 6 and 9 months in nondiabetic (ND) and streptozotocin-induced diabetic (STZ-D) rats. In ND rats, no difference in [11C]HED retention or norepinephrine content was detected in the proximal versus distal myocardium. After 6 months, compared with ND rats, myocardial [11C]HED retention had declined in the proximal segments of STZ-D rats by only 9% (NS) compared with a 33% decrease in the distal myocardium (P < 0.05). Myocardial norepinephrine content was similar in both ND and STZ-D rats. At 6 months, LV myocardial NGF protein content in STZ-D rats decreased by 52% in the proximal myocardial segments (P < 0.01 vs. ND rats) and by 82% distally (P < 0.01 vs. ND rats, P < 0.05 vs. proximal segments). By 9 months, [11C]HED retention had declined in both the proximal and distal myocardial segments of the STZ-D rats by 42% (P < 0.01 vs. ND rats), and LV norepinephrine content and NGF protein were decreased in parallel. Therefore, 6 months of STZ-induced diabetes results in heterogeneous cardiac sympathetic denervation in the rat, with maximal denervation occurring distally, and is associated with a proximal-to-distal gradient of LV NGF protein depletion. It is tempting to speculate that regional fluctuations of NGF protein in the diabetic myocardium contribute to heterogeneous cardiac sympathetic denervation complicating diabetes.