Conjugation of DNA fragments to protein carriers by glutaraldehyde: immunogenicity of oligonucleotide-hemocyanin conjugates

The practical realization of the concept of specific immunotherapy for systemic lupus erythematosus (SLE) has been hampered, thus far, by an inability to link DNA fragments to carrier protein. In this paper, a novel technique is described, in which glutaraldehyde is the linking agent. A 2-stage method was used to link oligonucleotides to a soluble protein carrier, such as keyhole limpet hemocyanin (KLH) or human gamma globulin (HGG), whereas a 1-stage technique was sufficient to link oligonucleotides to sheep red cells. Both the ultraviolet absorbance spectrum and diphenylamine assay demonstrated that oligonucleotides were coupled to soluble protein. The conjugate of oligonucleotide to protein carrier appears to be recognized by anti-DNA antibody since oligonucleotide linked to either KLH or HGG inhibited the binding of anti-DNA antibody in vitro, and oligonucleotide-coupled sheep cells are agglutinating by seropositve sera from lupus patients. In addition, oligonucleotide-KLH raised hemagglutinating antibody to denatured DNA in C57BL/6, DBA/2 or NZB mice, as well as IgG antibody as detected by SPRIA in C57BL/6 and DBA/2 mice. The significance of this new method for the development of an antigen specific therapy of SLE is discussed.     

Dopamine and methionine-enkephalin in human brain

The contents of dopamine (determined radioenzymatically) and methionine-enkephalin (assayed by a radioimmunoassay) were measured in several areas of the human brain. The peptide was principally localized in dopamine-rich structures. In patients with Parkinson's disease, in contrast to the general dopamine deficiency, the reduction in methionine-enkephalin was restricted to the mesencephalon, putamen and lateral pallidum.     

Intracytoplasmic and intranuclear Reinke's crystals in a testicular Leydig-cell tumor diagnosed by fine-needle aspiration cytology: a case report with review of the literature

We report on the cytopathologic findings of a Leydig-cell tumor of the testis in a young adult male with no evidence of endocrine dysfunction. The preoperative diagnosis was based on fine-needle aspiration cytology (FNAC) alone, which was subsequently confirmed on histopathology. The present case was of interest on account of the paucity of literature regarding the cytodiagnosis of this lesion. In addition, the finding of intracytoplasmic lipofuscin pigment and several intracytoplasmic as well as intranuclear Reinke's crystals served to clinch the diagnosis on FNA. Therefore, the use of FNAC, especially in the presence of diagnostic Reinke's crystals, may vitiate the need for more invasive biopsy procedures in the preoperative diagnosis of testicular Leydig-cell tumors.     

Screening for the two most frequent mutations in Leber's hereditary optic neuropathy by duplex PCR based on allele-specific amplification

This report describes a rapid and inexpensive assay, which allows detection, in whole blood and by PCR alone, of the two most frequent mitochondrial DNA mutations causing Leber's hereditary optic neuropathy. The assay is based on allele-specific amplification, using primers with the mutation-specific base in the 3′ position, and a deliberately introduced G→C Substitution of base no. four from the 3′ end, which prevents amplification of the wild-type allele. © 1993 Wiley-Liss, Inc.     

A method for the accurate determination of anti-hapten cytotoxic T lymphocyte precursors: correction for apparent 'anti-self' reactivity

A method is described for accurately determining the frequency of precursors of hapten specific cytotoxic T cells. The method is based on a standard Poisson analysis of limit dilution cultures, but makes a correction of 'anti-self' reacting clones and for spontaneously arising clones that recognise modified self. These corrections are shown to be especially important when low hapten densities are used, where there may be more than a 10-fold difference between the corrected and uncorrected frequency estimates. Determined levels of antigen specificity and of H-2 restriction are significantly enhanced by application of this method.     

Analysis of the Equalization of Inverted Repeats and Neurovirulence Using a Pseudorabies Virus Mutant Strain Altered at the Ul/Ir Junction

A recombinant pseudorabies (Aujeszky’s disease) virus (PrV) designated as vE16lac was constructed by deleting a 3-kbp DNA segment spanning the junction of long and short components of the viral genome, and by replacing the deleted segment with a lacZ-expression cassette. The aim of constructing this mutant was (a) to determine whether the terminal repeat (Tr) can serve as a template for the regeneration of the internal repeat (Ir), and (b) whether this deletion causes a reduction in the neuroinvasiveness of the virus. To analyze the mechanism of equalization, revertant viruses were selected and structurally characterized from vE16lac infection of PK-15 cells, mice and pigs. Because all revertants acquired Ir sequences identical to that of the wild-type virus, the equalization process occurred using the Tr as a template to reconstitute the Ir. We also found that the recombinant virus vE16lac was virulent in both pigs and mice. The data are discussed in view of studies performed with similar PrV mutants by other authors (Rall et al., 1992, Dean and Cheung, 1995 and Dean et al., 1996). This revised version was published online in July 2006 with corrections to the Cover Date.     

IL-8 as an important chemoattractant for neutrophils in ulcerative colitis and Crohn's disease.

IL-8 is generating increasing interest as a powerful neutrophil chemoattractant and activator. To elucidate the mechanisms of neutrophil infiltration in inflammatory bowel disease, we examined 33 patients with ulcerative colitis (UC), 18 with Crohn's disease (CD), eight with some other type of colitis, and 18 normal control subjects for measurement of IL-8 in homogenates of colonic biopsy specimens. The affected colonic mucosa was found to contain significantly more IL-8 in patients with active inflammatory bowel disease than in patients with inactive disease (UC, P < 0.001; CD, P < 0.001), in patients with other types of colitis (UC, P < 0.05; CD, P < 0.01), or in normal control subjects (UC, P < 0.001; CD, P < 0.001). Colonic IL-8 levels correlated significantly with the macroscopic grade of local inflammation, especially in patients with UC (P < 0.001). Colonic IL-8 levels also correlated well with the neutrophil numbers in mucosal tissue (UC, r = 0.950, P < 0.001; CD, r = 0.940, P < 0.001), and with colonic IL-1 beta (r = 0.911, P < 0.001) and tumour necrosis factor-alpha (TNF-alpha) levels (r = 0.604, P < 0.001) in patients with these two conditions. These data suggest a potential role for IL-8 and its regulatory cytokines IL-1 and TNF-alpha in mediating neutrophil infiltration of the gut wall in inflammatory bowel disease.     

Ultrastructural responses by Golgi apparatus of rat submandibular gland to beta-adrenergic, alpha-adrenergic, and cholinergic stimulation

Rat submandibular gland tissue pieces were stimulated in vitro for 30 min with a beta-adrenergic agent or a cyclic AMP analog to stimulate protein secretion, or with alpha-adrenergic or cholinergic agents or a Ca2+ ionophore to stimulate fluid secretion. Acinar cells were examined by transmission electron microscopy. In control tissue, acinar cells showed little evidence of secretory activity. The Golgi apparatus was sparse and was associated with a few small, immature secretory granules with fine fibrillar contents. Following secretory granule discharge stimulated by isoproterenol or dibutyryl cyclic AMP, acinar cells were constricted, and had extensive basolateral membrane folding and tightly packed rough endoplasmic reticulum. Golgi complexes were prominent and had multiple small granules with filamentous contents. After stimulation of fluid secretion by alpha-adrenergic agents (epinephrine, phenylephrine), or cholinergic agents (acetylcholine, carbachol, pilocarpine), or a Ca2+ ionophore (A23187), the Golgi apparatus had compact concave cisternae enclosing aggregates of tubulovesicles. Acinar cells were distended, basolateral membranes were expanded, and rough endoplasmic reticulum was dilated and vesiculated.     

Shared allergenic and antigenic determinants in Alternaria and Stemphylium extracts

To develop a model for mold allergen extract standardization, we studied eight commercial Alternaria extracts from various suppliers by a variety of immunochemical and physicochemical techniques, including measurement of Alt-I, a purified allergenic fraction of Alternaria. Wide variations were noted in the allergenic and antigenic potencies of these extracts. Estimates of Alt-I content measured by Alt-I RAST inhibition and by radioimmunoassay correlated significantly (p < 0.05), but Alt-I activity by either method could not be correlated with allergenic potency as measured by RAST inhibition using solid-phase Alternaria. Each test extract produced unique and differing patterns of Coomassie blue-stained bands in isoelectrofocusing gels and in crossed immunoelectrophoresis gels using rabbit antibodies to Alternaria. The optimal method for mold allergen standardization involves a combination of RAST inhibition, isoelectrofocusing, and crossed immunoelectrophoresis techniques, and, if possible, quantitation of individual allergens.