Measurement of platelet P-selectin for remote testing of platelet function during treatment with clopidogrel and/or aspirin.

There is great interest in assessing the efficacy of treatment with clopidogrel and aspirin in patients with cardiovascular disease using procedures that can be used in a remote setting. Here we have established methods to assess the effects of clopidogrel and aspirin on platelets based on measurements of platelet P-selectin. Platelets were stimulated in whole blood by adding the combination of adenosine diphosphate and the TXA(2) mimetic U46619 (ADP/U4, designed to assess P2Y(12) inhibition) or the combination of arachidonic acid and epinephrine (AA/Epi, designed to assess COX-1 inhibition). The stimulated samples were then fixed using a fixative solution that provides stability for at least 9 days, and sent to a central laboratory for analysis of P-selectin by flow cytometry. Measurements were performed in blood from healthy volunteers and patients with cardiovascular disease. The inhibitory effects of clopidogrel and aspirin were assessed ex vivo and the effects of the direct acting P2Y(12) antagonist cangrelor and aspirin were assessed in vitro. Measurements of platelet aggregation were also performed for comparison. In healthy volunteers clopidogrel ex vivo and cangrelor in vitro markedly inhibited P-selectin expression induced by ADP/U4. Aspirin did not inhibit and did not interfere with the effects of clopidogrel or cangrelor using this test. There was very little overlap of results obtained in the absence and presence of clopidogrel or cangrelor. In contrast, over half of 42 patients with cardiovascular disease did not respond well to clopidogrel treatment, although cangrelor was still effective. Aspirin markedly inhibited P-selectin expression induced by AA/Epi. Clopidogrel had much less effect and did not interfere with the effects of aspirin. There was no overlap of results obtained in the absence and presence of aspirin. Aspirin provided near-complete inhibition in 29 of 30 patients with cardiovascular disease. Aggregometry measurements agreed well with the P-selectin data obtained ex vivo following both clopidogrel and aspirin treatment. It is concluded that measurements of P-selectin performed on fixed blood samples following platelet stimulation in whole blood in a remote setting can be used effectively to monitor the effects of clopidogrel and aspirin.

Avidity determination of Borrelia burgdorferi-specific IgG antibodies in Lyme disease.

The avidity indices of Borrelia burgdorferi-specific IgG antibodies were estimated using ELISA in sera from patients with different stages of Lyme disease. In addition, sera from healthy students with proof of borrelial-specific IgG antibodies from standard serology were tested. Low avidity indices were detected predominantly in sera from patients with early-stage Lyme disease [erythema migrans (EM); n = 25]. High avidity indices were found in healthy students (n = 72) and in most of the patients with neuroborreliosis (NB; n = 44) and chronic late-stage Lyme disease [acrodermatitis chronica atrophicans (ACA); n = 36]. In conclusion, early-stage Lyme disease (EM) could be differentiated from advanced and chronic stages (NB, ACA) and from "seropositive" healthy persons using avidity determination in the majority of patients in this study.

Esophagobronchial fistula combined with a peptic esophageal stenosis.

Peptic strictures are a rare complication of severe gastroesophageal reflux disease. An esophagobronchial fistula as a complication of a severe long-term reflux esophagitis with peptic stenosis is here described for the first time: A 43-year-old mentally disabled patient suffered from recurrent bronchopneumonia. Endoscopy revealed an esophagobronchial fistula originating in a peptic stricture. Under short-term fasting, intravenous feeding and application of a proton pump inhibitor (PPI) closure of this fistula was achieved within 4 days. Subsequently, dilatation was carried out. The case demonstrates that pulmonary complications in patients with peptic esophageal strictures may not only be due to aspiration of refluxate but--rarely--also to fistulae between the esophagus and the bronchial tree.

Bacteriologic and immunologic aspects of gram-negative folliculitis: a study of 46 patients.

BACKGROUND: Gram-negative folliculitis is an infection with gram-negative rods that most often occurs as a complication of prolonged broad-spectrum antibiotic therapy in patients suffering from acne and rosacea. METHODS: The bacteriologic and immunologic findings are reported in 46 patients, 39 men and 7 women, aged 16-79 (median, 28) years, with gram-negative folliculitis. Hypersensitivity reactions to various microbial recall antigens as well as granulocyte functions were evaluated. Quantitative measurements of serum levels of immunoglobulin M, G, A, and E, total complement activity, complement factors C3 and C4, and alpha-1-antitrypsin were performed. RESULTS: The gram-negative organisms most frequently cultivated from nares, facial skin, and pustules were Klebsiella spp., Escherichia coli, Enterobacter spp., and Proteus spp. In all patients, deviations of one or more immune parameters were detected, including lowered serum concentrations of immunoglobulin M and alpha-1-antitrypsin, and elevated levels of immunoglobulin E. The humoral and cellular parameters were not influenced by isotretinoin therapy of gram-negative folliculitis. CONCLUSIONS: These findings suggest that gram-negative folliculitis is not only a complication of long-lasting antibiotic treatment of acne and rosacea, but might be an entity of its own. Immunologic factors may play a critical role in the pathogenesis of gram-negative folliculitis.

Analysis of antibody response to the outer surface protein family in lyme borreliosis patients.

Studies on frequencies of serum antibodies to outer surface proteins (Osps) in Lyme disease have produced conflicting results. Osp antigens (A, B, and C) enriched by butanol extraction, which aids band identification in immunoblotting, were used to test sera for IgG antibody to Osp antigens from Borrelia burgdorferi isolates from each subspecies (sensu stricto, afzelii, and garinii). Individual isolates were selected to include all five known European OspA genotypes. Of arthritis sera, 83% (n=29), and of acrodermatitis chronica atrophicans sera, 81% (n=26), recognized OspA, B, and/or C. Of erythema migrans sera, 23% recognized OspA and/or B and a further 15% OspC alone. Only 5 (6%) of 86 sera (4 arthritis, 1 acrodermatitis chronica atrophicans, 0 erythema migrans) recognized all five OspA phenotypes tested. Marked differences in the reactions of individual sera to the various Osp antigens were seen, which helps reveal the causes of discrepancies between previous reports.

Current antimicrobial susceptibility of cutaneous bacteria to first line antibiotics.

Antimicrobial susceptibility of common bacterial species occurring on human skin appears to be falling. Data for the antimicrobial susceptibility of major groups of bacteria isolated from human skin during routine cultures were complied and analysed over a period of 9 months. Routine diagnostics of specimens from skin lesions and normal human skin were analysed for the presence of specified groups of bacteria. The species were identified using standard methods. Anti-microbial susceptibility was determined using a broth microdilution system giving breakpoints, the Sensititre system. Of the 333 Staphylococcus aureus, 129 Streptococcaceae, 180 Enterobacteriaceae and 120 Pseudomonadaceae strains investigated more than 5% of Staphylococcus aureus strains were resistant to flucloxacillin and thus methicillin (MRSA). More than 25% of Staphylococcus aureus strains were resistant to tetracycline and erythromycin. Many MRSA strains were found multi-resistant. Gentamicin was active against a large majority of Enterobacteriaceae strains but many Pseudomonadaceae strains were resistant. Compared with previous corresponding surveys methicillin-resistant Staphylococcus aureus strains are clearly on the increase. To prevent a further increase of resistant strains a defined strategy for antibiotic use is needed in dermatology.

Enzyme-linked immunosorbent assay using recombinant OspC and the internal 14-kDa flagellin fragment for serodiagnosis of early Lyme disease.

The outer surface protein C (OspC) and the internal 14-kDa flagellin fragment of strain GeHo of Borrelia burgdorferi sensu stricto were expressed as recombinant proteins in Escherichia coli and were purified for use in an immunoglobulin M (IgM) enzyme-linked immunosorbent assay (OspC-14-kDa antigen ELISA). No hint at disturbing protein-protein interferences, which might influence the availability of immunoreactive epitopes, was found when the recombinant antigens were combined in the ELISA. The recombinant OspC-14-kDa antigen ELISA was compared to a commercial IgM ELISA that used a detergent cell extract from Borrelia afzelii PKo as the antigen. According to the manufacturer's information, the cell extract contains, in addition to other antigens, the following diagnostically relevant antigens: the 100-kDa (synonyms, 93- and 83-kDa antigens), 41-kDa, OspA, OspC, and 17-kDa antigens. The specificity was adjusted to 95% on the basis of data for 154 healthy controls. On testing of 104 serum samples from patients with erythema migrans (EM), the sensitivity of the recombinant ELISA (46%) for IgM antibodies was similar to that of the commercial ELISA (45%). However, when 42 serum samples from patients with polyclonal B-cell stimulation due to an Epstein-Barr virus infection were tested, false-positive reactions were significantly less frequent in the recombinant ELISA (10%) than in the whole-cell-extract ELISA (23%). OspC displays sequence heterogeneity of up to 40% according to the genomospecies. However, when the reactions of serum specimens from controls and EM patients with OspC from representative strains of B. burgdorferi sensu stricto (strain GeHo) and B. afzelii (strain PKo) were compared in an ELISA, almost no differences in specificity and sensitivity were seen. This demonstrates that the sera predominantly recognize the common epitopes of OspC tested in this study. In conclusion, we suggest that the OspC-14-kDa antigens ELISA is a suitable test for the detection of an IgM response in early Lyme disease.

Establishment of enzyme-linked immunosorbent assay using purified recombinant 83-kilodalton antigen of Borrelia burgdorferi sensu stricto and Borrelia afzelii for serodiagnosis of Lyme disease.

The 83-kDa antigen of Borrelia burgdorferi was expressed as a recombinant protein in Escherichia coli and purified for use in an enzyme-linked immunosorbent assay (p83-ELISA). Antibodies to the 83-kDa antigen of both the immunoglobulin G (IgG) and IgM isotypes could be detected in all stages of Lyme disease. Sensitivity varied, depending on the clinical stage of illness. In early stages, as defined for 118 patients with erythema migrans, it was found to be 20% (24 of 118 patients: 7 with IgM, 16 with IgG, and 1 with IgM and IgG). Of the patients with late-stage Lyme arthritis and acrodermatitis chronica atrophicans, 94% (16 of 17:2 with IgM and IgG and 14 with IgG) and 86% (36 of 42:2 with IgG and IgM and 34 with IgG) revealed positive results in the p83-ELISA, respectively. p83 displays sequence heterogeneity according to the genomospecies, but when the reactions of serum specimens from acrodermatitis chronica atrophicans patients and arthritis patients with p83 derived from representative strains of B. burgdorferi sensu stricto and Borrelia afzelii in ELISAs were compared, no differences in specificity and sensitivity were seen. When 82 serum specimens from healthy controls were tested, none had IgG and only 3 (4%) had IgM antibodies, indicating a high specificity. Positive reactions with antibodies against Treponema pallidum (1 of 37 patients; IgG) and Epstein-Barr virus (1 of 44 patients; IgM) and with autoantibodies of various specificities (1 of 53 patients; IgG) were seen with < 3% of the serum samples te11111111111111111111 high speficicity for B. burgdorferi.2+ 13% for IgM antibodies, the IgM p83-ELISA provided little diagnostic information for Lyme disease, whereas the IgG p83-ELISA appears to be a suita ;e test for serodiagnosis of advanced-stage Lyme disease.