A differential effect of C5a and C5a des Arg in the induction of pulmonary inflammation.

Earlier studies have shown that C5 fragments induce an inflammatory reaction when instilled into the rabbit lung. Because C5a is rapidly converted to C5a des Arg in vivo, experiments were performed to determine which fragment was most effective in producing pulmonary inflammation in this animal model. C5a des Arg consistently produced marked inflammation. This was characterized by neutrophil accumulation, edema, hemorrhage, fibrin formation, and damage to alveolar epithelium. The time course of the inflammatory reaction initiated by C5a des Arg showed pulmonary vascular sequestration of neutrophils with no intra-alveolar migration at 30 minutes after injection. By 2 hours, interstitial and alveolar neutrophils were numerous, with the accumulation of neutrophils in the alveoli increasing to a maximum at 6 hours. At 24 and 48 hours, the predominant cells were mononuclear (macrophages). By 120 hours, the lesions were resolving. In contrast, at all doses examined, a similar instillation of C5a induced either no inflammation or a milder, more focal response than C5a des Arg. This inability of C5a to initiate inflammation was not apparently due to the generation of inhibitors, since mixtures of C5a and C5a des Arg were phlogistic. A prolonged, intrapulmonary infusion of C5a (20 minutes), in contrast to a bolus instillation (1 minute), did initiate an inflammatory response, which may reflect the conversion of the C5a to C5a des Arg in the lung. This study points out the inflammatory potential of products of complement activation, particularly of the C5 fragment C5a des Arg, when applied to the airway side of the lungs. This inflammatory response raises the possibility that cleavage of intrapulmonary C5 may play an important role in the initiation of pulmonary inflammation.     

Human skin fibroblast in vitro tetraploidy. Flow cytometric DNA assay used to confirm metaphase assay in patients with various colonic diseases

One hundred twenty-two patients with various colon pathologies (43 colorectal carcinoma patients exclusive of the known autosomal dominant colorectal cancer syndromes, 31 patients with solitary colorectal adenomas, 27 patients with ulcerative colitis, and 21 controls with no evidence of colorectal cancer) were investigated for in vitro tetraploidy in dermal fibroblasts cultures using a metaphase assay for determination of numerical chromosomal alterations. Later, stationary cultures of these skin fibroblasts were investigated with DNA flow cytometry. There was good correlation between the percentage of tetraploid metaphase cells and the percentage of nuclei with a flow cytometric DNA index of 2. Using a linear discrimination function to classify the flow cytometric data, the peak with DNA index of 2 was the most important parameter, supplemented by the region with DNA index greater than 2, whereas the region with DNA index between 1 and 2 probably represented a different subpopulation. We have thus demonstrated that only the region with a DNA index of 2 or greater is suitable in determining in vitro tetraploidy in stationary skin fibroblast cultures.     

Alignment of the restriction map of mouse adenovirus FL with that of human adenovirus 2

A detailed restriction map of mouse adenovirus (AdFL) has been constructed based on sites of cleavage of AdFL DNA by 19 restriction endonucleases. The AdFL map has been oriented with that of human adenovirus 2 (Ad2) by identifying which end of AdFL DNA is retained in virus particles with incomplete genomes compared with the end retained by Ad2 defective particles (C. Tibbetts, 1977, Cell12, 243–249). The two maps were also oriented by cross-hybridization tests with a series of restriction fragments of each viral DNA. The latter experiments revealed two regions of homology between Ad2 and AdFL DNA, corresponding to the positions of the Ad2 hexon gene and an Ad2 hexon-associated protein gene.     

Identification of a type 1 diabetes-associated CD4 promoter haplotype with high constitutive activity

CD4 is a candidate gene in autoimmune diseases, including Type 1 diabetes mellitus (T1DM), because the CD4 receptor is crucial for appropriate antigen responses of CD4(+) T cells. We previously found linkage between a CD4-1188(TTTTC)(5-14) promoter polymorphism and T1DM. In the present study, we screened the human CD4 promoter for mutations and identified three frequent single nucleotide polymorphisms (SNPs): CD4-181C/G, CD4-521C/G and CD4-1050T/C. The SNPs are in strong linkage disequilibrium (LD) and association with the CD4-1188(TTTTC)(5-14) alleles, and we observed nine CD4 promoter haplotypes, of which four are frequent. We genotyped the SNPs in 253 Danish T1DM families (1129 individuals) and found evidence for linkage and association of a CD4 (A4(-1188)T(-1050)G(-521)C(-181)) haplotype to T1DM. In reporter studies, we show that (1) the T1DM-associated CD4 haplotype encodes high constitutive promoter activity and (2) the CD4-181G variant encodes higher stimulated promoter activity than the CD4-181C variant. This difference is in part neutralized in the frequently occurring CD4 promoter haplotypes by the more upstream genetic variants. Thus, we report functional impact of a novel CD4-181C/G SNP on stimulated CD4 promoter activity and the identification of a novel CD4 haplotype with high constitutive promoter activity that is linked and associated with T1DM.     

Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler

During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism.     

Acute and Subacute Toxicity of Aspilia Africana Leaves

This study was designed to evaluate the toxicity of the aqueous extract of Aspilia africana leaves. Oral doses of 500 mg/kg and 1000 mg/kg were administered for 28 days to rats after every 2 days for sub-acute toxicity. For acute toxicity, 5 doses of 2, 4, 8, 12 and 16g/Kg body weight were investigated in mice. The control groups consisted of mice or rats administered with distilled water. The signs of toxicity fluctuated lightly from one mammal to another throughout the experiment. The liver, kidneys and heart weight of rats revealed no significant differences between the test groups and the control. The results indicated that the medium lethal dose (LD₅₀) was found to be greater in females than males with an average of 6.6g/Kg body weight for both sexes. Regardless of the significant differences observed at certain points in some biochemical parameters (ALT, AST, ALP, Creatinine and Glutathione); none showed any linear dose responsiveness. On the other hand, most of the parameters investigated were found to be gender dependent. These results suggested that A Africana can be classified among substances with low toxicity.     

Humoral Immunity to Dietary Antigens in Atopic Dermatitis

Enzyme-linked immunosorbent assays (ELISA) employing a biotin-avidin amplification step are described for the quantification of human serum IgG antibodies to the dietary antigens ovalbumin (OA) and beta-lactoglobulin (BLG). The analytical quality of these assays was acceptable. Antibodies were measured in 16 patients with mild or moderate atopic dermatitis (AD), in 31 patients with a history of AD, and in closely matched controls. Levels of serum anti-OA antibodies did not differ in patients and controls, whereas anti-BLG antibodies tended to be higher in patients with mild or moderate AD than in controls (P less than 0.05).     

Problems affecting performance of the fluorescent treponemal antibody-absorption test for syphilis.

Immunofluorescent staining of Treponema pallidum was studied to clarify the effect of three factors on the results of the fluorescent treponemal antibody-absorption test: (i) heat inactivation of sera at 56 degrees C for 30 min before testing, (ii) use of multicircle slides, and (iii) tungsten illumination to visualize and assess unstained treponemes on reactive as well as nonreactive smears. It was found that serum inactivation before testing was not necessary for detection of immunoglobin G antibody, but an immunoglobulin M prozone was detected in unheated serum. On multicircle slides, it was demonstrated that a false-positive reaction could be obtained in 30 s at 37 and 25 degrees C if a smear where a nonreactive serum had been placed was crossed by a strongly reactive serum from another circle. Tungsten illumination proved necessary for correct assessment of unstained treponemes on all fluorescent treponemal antibody-aborption test smears, reactive or nonreactive. The possible role of these factors in incorrect fluorescent treponemal antibody-absorption test results is discussed.