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The present clinical study aimed to investigate if there are differences in microbiological outcomes dependent on the subgingival biofilm collection method. Subgingival biofilm samples were collected from the four deepest pockets (>5 mm) of 17 patients with aggressive periodontitis (AgP) and 33 patients with chronic periodontitis (CP), first by paper point and thereafter by curette. Samples obtained with the same method were pooled together from each patient and forwarded for molecular microbiological analysis by a commercially available assay (IAI Pado Test 4.5) that estimates total bacterial load and levels of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and Aggregatibacter actinomycetemcomitans. Data analysis included frequency of detection, quantification and correlation of detection levels between the two sampling methods. P. gingivalis, T. forsythia and T. denticola were detected in >90% of the samples, and their detection levels exhibited a strong correlation between sampling methods. The detection consistency of A. actinomycetemcomitans was 56% between the two sampling methods. A. actinomycetemcomitans was more readily detected by paper point compared with curette collection with a stronger correlation between the two methods in AgP. Subgingival biofilm sampling by curette or paper point does not yield differences in the detection of the three 'red complex' species. However, A. actinomycetemcomitans was more consistently detected by means of paper point collection, which can be crucial in the decision to administer antibiotics as an adjunctive periodontal treatment., (© 2013 APMIS. Published by John Wiley & Sons Ltd.)

Paper points are widely established for the collection of subgingival plaque or other oral samples to analyze the microbiota, especially the presence of periodontal pathogenic bacteria such as Actinobacillus actinomycetemcomitans or Porphyromonas gingivalis. In contrast to the high frequency of usage of paper points in oral sampling, very few data are available about the parameters influencing the sampling process. Therefore, in four different in vitro experiments (6-9 repeats), we inoculated paper points with standardized suspensions (2 x 10(9) colony-forming units/ml) of A. actinomycetemcomitans and P. gingivalis to test the influence of the origin (kind) of paper point ("manufacturer"), size (according to the International Organization for Standardization ISO 25-80), sampling time (5 to 60 s), and elution time (5 to 60 s). Sampled bacteria were detected and (semi-) quantified using 16S rRNA/DNA-directed oligonucleotide probes. The bacterial load was categorized and calculations performed with index values ranging between 0 (below the detection limit of 10(2) to 10(3) colony-forming units) and 9 (> 10(6) colony-forming units). We found statistically significant differences in the efficiency of bacterial sampling between the 5 paper point manufacturers tested, expressed in a mean bacterial index between 4.4 and 7.8. ISO 45 Paper points were statistically proven to work most efficiently. According to our results, a sampling time of 60 s seems to be optimum. However, shorter times between 5 and 30 s did not significantly reduce the sampling efficiency. We found an interval of 20 s best to elute bacteria from the paper points by vortexing. The evaluation of basic parameters for subgingival plaque sampling by paper points might help to optimize microbiologically based diagnostics in periodontal diseases.

We have experimentally immunized rats intratracheally with sonicated Pseudomonas aeruginosa to develop a high antibody response systemically and locally. The systemic antibodies were measured by a standard enzyme-linked immunosorbent assay (ELISA) on serum samples, whereas the local antibodies were measured on eluates from paper discs containing saliva. High levels of IgA antibodies were elicited in saliva whereas only traces of IgG antibodies were detected; the opposite results were found in serum. The saliva disc method permits serial measurements from the same animal and therefore offers the possibility to follow post-immunization antibody responses.

The production and partial characterization of monoclonal hybridoma antibodies against Mycobacterium tuberculosis is described. 30 clones have been produced. Their reactivity towards M. tuberculosis antigens have been tested in immunoblotting from SDS-PAGE, in crossed immunoelectrophoresis and in ELISA. The Mab's separated into four reactivity groups, one of which may be relevant in assays for antigen specific for M. tuberculosis.

The hepatitis B surface antigen (HBsAg) is highly immunogenic and induces an antibody response which is protective in vivo against hepatitis B virus (HBV) infection. Human monoclonal antibodies specific for HBsAg were produced, which could have potential therapeutic applications. Lymphocytes obtained from a vaccinated donor were stimulated in vitro and fused with the human myeloma cell line GM 4672, and eight hybridomas were obtained. Three of these clones, which reacted in an ELISA against the HBsAg vaccine, were expanded, subcloned and further analyzed. The subclones E7C2, C4C10, and D5B2 were able to bind to different HBsAg preparations, which express various subtypes, and recognized the major HBsAg peptides in Western blot analysis. Cross-inhibition experiments showed that E7C2, C4C10 and D5B2 are directed against the same epitope and have an affinity constant ranging from 5 X 10(7) to 3.3 X 10(9) M. Furthermore, these antibodies stained the surface and cytoplasm of the HBsAg-secreting cell lines PLC/PRF/5 and 4.10. The production of immunoglobulins varies from 0.3 to 1.3 micrograms/ml/10(6) and has remained stable over a period of 8 months. These human monoclonal antibodies, which appear to be directed against an antigenic determinant common to all HBsAg subtypes, could be useful in the study of HBV-related liver diseases as well as in their diagnosis and experimental therapy.

The distribution of NK-9 antigen presenting cells in human lymphoid tissues including spleen, tonsil, lymph node, and thymus was studied. The staining were done on formalin-fixed, histological sections. In peripheral blood NK-9 antigens are expressed on allospecific (CTL) and nonspecific (NK and AK) cytotoxic lymphocytes. In lymphoid tissues, the distribution of NK-9 positive cells was definitely different from "normal" T cell distribution, measured by pan T monoclonal markers. In tonsils and lymph nodes NK-9 positive cells were mainly located in and around germinal centers. However, only a proportion of these areas were NK-9 positive. In spleen NK-9 positive cells were located mainly in periarteriolar lymphocyte sheats and in germinal centers. Thymuses were practically NK-9 negative. These results support the earlier suggestions that cytotoxic cells take part in the immunoregulation in lymphoid tissues.