A wide spectrum of fastidious and ampicillin-susceptible bacteria dominate in animal-caused wounds.

The main purpose of this study was to assess the actual occurrence of Gram-negative oxidase-positive bacteria (GNOP) in human wounds caused by animals, mostly cat and dog bites and scratches, and with signs of infection. We report a prospective series of 92 wound samples. Routine culturing was combined with a procedure optimised for fastidious GNOP. All GNOP isolates were identified by 16S rDNA sequencing to the species level. We observed a more prominent role of GNOP, including at least 30 species mostly in the families Flavobacteriaceae, Neisseriaceae and Pasteurellaceae, and less of Staphylococcus aureus and streptococci. The antibiotic susceptibility pattern was investigated, as GNOP are associated with sudden onset of serious infections, making an early decision on antibiotic treatment vital. All GNOP isolates judged to be clinically relevant displayed susceptibility to ampicillin and meropenem, but resistance to oxacillin, clindamycin and gentamicin was frequent. Our findings emphasise the need to cover GNOP as recommended in guidelines, and not only common wound pathogens, when treating an animal-caused wound.

Heterogeneity among septic shock patients in a set of immunoregulatory markers.

Immune activation is a regular feature of sepsis, but the incidence and nature of the ensuing inflammation-resolving and immunosuppressive component is less well understood. In this study, we compared immunoregulatory markers on blood leukocytes from patients with Gram-negative or Gram-positive sepsis or septic shock, and compared this to blood from patients with severe virosis or healthy controls. To this end, blood from 32 patients with sepsis, including ten cases with shock, and 12 patients with severe virosis were analysed by flow cytometry for the expression levels of monocyte HLA-DR, CD11c, CD14 and CD40, and for frequencies of CD163(+)-suppressive monocytes, HLA-DR(+) or CD40(+)-activated T cells and Tregs. Plasma cytokine levels were analysed as a functional measurement. Signs of immunosuppression dominated in the septic shock and Gram-positive sepsis groups, whereas monocyte activation was common in Gram-negative sepsis patients without shock. However, the main finding was the large inter-individual variation of immune activation and immunosuppression, with no correlation to prognosis among the shock patients. The pronounced inter-individual variation in the analysed monocyte and lymphocyte markers forms a strong argument that, when immunomodulatory treatment is considered in a sepsis patient, it should be personalised and guided by a detailed immune status assessment.

Wnt5a inhibits human monocyte-derived myeloid dendritic cell generation.

Wnt5a is a non-canonical Wnt protein that is expressed at elevated levels in inflammatory conditions. Its role in inflammation remains unclear, although it is known that Wnt5a is expressed at a higher level in monocyte-derived myeloid dendritic cells (Mo-mDCs) than in monocytes and macrophages. The function of Wnt5a in dendritic cells (DCs) remains relatively unexplored. Here, we found that under Mo-mDC culture conditions, Wnt5a inhibited the generation of CD14(⁺/low) Mo-mDCs while promoting the generation of CD14⁺/⁺⁺ CD16⁺ monocytes. We could further show that stimulation of monocytes with rWnt5a induced a rapid IL-6 production and that the rWnt5a treated Mo-mDC differentiation was restored upon blocking of IL-6. Also, conditioned media from Wnt5a stimulated human breast cancer cells producing IL-6, specifically inhibited Mo-mDC differentiation. These observations are strengthened by our finding that patients with sepsis, a disease involving elevated Wnt5a and IL-6 levels, also showed a significant increase in the CD14⁺ CD16⁺⁺/CD14⁺/⁺⁺ CD16⁺ monocyte populations, which was accompanied by a significant decrease in circulating mDCs. We finally show that under typical Mo-mDC culture conditions, monocytes isolated from patients with sepsis as compared to healthy controls, preferentially differentiated into CD14CD14⁺/⁺⁺ HLA-DR⁺⁺ cells. We suggest that Wnt5a is a possible candidate mediator for the CD14⁺/⁺⁺ CD16⁺ monocyte accumulation seen in patients with infectious disease and cancer.

TOF-SIMS analysis of exhaled particles from patients with asthma and healthy controls.

Particles in exhaled air (PEx) may reflect the composition of respiratory tract lining fluid (RTLF); thus, there is a need to assess their potential as sources of biomarkers for respiratory diseases. In the present study, we compared PEx from patients with asthma and controls using time-of-flight-secondary ion mass spectrometry (TOF-SIMS) and multivariate analysis. Particles were collected using an instrument developed in-house. 15 nonsmoking subjects with physician-diagnosed asthma and 11 nonsmoking healthy controls performed 10 consecutive forced exhalations into the instrument. Particle concentrations were recorded and samples of particles collected on silicon plates were analysed by TOF-SIMS. Subjects with asthma exhaled significantly lower numbers of particles than controls (p=0.03) and the ratio of unsaturated to saturated phospholipids was significantly lower in samples from subjects with asthma (0.25 versus 0.35; p=0.036). Orthogonal partial least squares-discriminant analysis models showed good separation between both positive and negative spectra. Molecular ions from phosphatidylcholine and phosphatidylglycerol, and protein fragments were found to discriminate the groups. We conclude that analysis of PEx is a promising method to examine the composition of RTLF. In the present explorative study, we could discriminate between subjects with asthma and healthy controls based on TOF-SIMS spectra from PEx.

Hereditary dysplastic nevus syndrome: lymphoid cell ultraviolet hypermutability in association with increased melanoma susceptibility.

The hereditary dysplastic nevus syndrome (DNS) is a well-characterized disorder in which affected individuals have increased numbers of premalignant (dysplastic) nevi and a markedly increased risk of developing cutaneous melanoma. Seeking evidence of a systemic disorder in DNS, we examined the effect of ultraviolet radiation on cultured lymphoid cells. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS had similar survival values following treatment with 2.3 to 9.0 J of 254-nm ultraviolet radiation per m2 as did lines from control individuals. Mutagenesis at the hypoxanthineguanine phosphoribosyltransferase locus was assessed by measuring the induction of resistance to thioguanine using a microtiter well assay. Three lymphoblastoid cell lines from patients with hereditary DNS and melanoma had a 2- to 3-fold greater frequency of induced mutants per clonable cell than three normal lines following exposure to 4.5 to 9.0 J of ultraviolet radiation per m2. Expanded clones of mutated DNS lymphoblastoid cell lines had less than 6% of normal hypoxanthine-guanine phosphoribosyltransferase activity. Inhibition and recovery of DNA synthesis following ultraviolet exposure were similar in 2 DNS and 2 normal lines. Repair by DNS lines of ultraviolet-induced DNA damage was in the normal range as measured by alkaline elution. Thus, hereditary DNS exhibits in vitro hypermutability which may reflect increased susceptibility to ultraviolet-induced somatic mutations in vivo. This abnormality may be related to the increased melanoma susceptibility of patients with hereditary DNS.

Triple helix directed psoralen adducts induce a low frequency of recombination in an SV40 shuttle vector.

Triple helix forming oligonucleotide directed psoralen adducts in a mammalian shuttle vector have been reported to be repaired efficiently in human cells. In this study we examined the role of intermolecular homologous recombination in triple helix targeted psoralen adduct repair. A simian virus 40 (SV40) shuttle vector carrying a mutated supF gene was treated with a triplex forming oligonucleotide psoralen conjugate and cotransfected into human cells with a second plasmid bearing the wild type supF gene. Recombinants with a reactivated marker gene were detected by an X-gal assay in indicator bacteria. We could observe a low frequency of psoralen adduct induced recombination indicating that recombination does not play a major role in triplex directed psoralen adduct repair. The implications for targeted mutagenesis by triple helix forming oligonucleotides are discussed.

Normal rate of DNA breakage in xeroderma pigmentosum complementation group E cells treated with 8-methoxypsoralen plus near-ultraviolet radiation.

Treatment of normal and xeroderma pigmentosum complementation group E skin fibroblasts with 8-methoxypsoralen plus repeated doses of near-ultraviolet radiation elicited a marked increase in DNA strand breakage during a subsequent incubation. No such induction of breaks was noted with cells from xeroderma pigmentosum groups A and D. The results suggest that the gene product which is deficient in xeroderma pigmentosum group E cells is involved in a critical step of DNA repair of far-ultraviolet photoproducts but not so in the repair of psoralen cross-links.

Ku protein in human T and B lymphocytes: full length functional form and signs of degradation.

DNA-dependent protein kinase (DNA-PK) has been shown to take part in cell cycle regulatory signal transduction and in the repair of X-ray-induced DNA double-strand breaks. Functional DNA-PK is furthermore needed for the generation of antigen specificity during lymphocyte maturation. The Ku86 subunit of DNA-PK has been reported to exist in human B lymphocytes in a truncated form capable of binding to broken DNA but lacking the ability to activate the kinase function of DNA-PK. In the present work the Ku70 and Ku86 dimer proteins in T and B lymphocytes from human blood donors were analysed by immunoblotting and were observed apparently to be of full length. Also, nuclear protein extracted from B and non-B lymphocytes displayed DNA-dependent kinase activity. However, a minor fraction of Ku86 in lymphocytes was observed to be truncated with a molecular mass of approx. 70 kDa.

DNA damage in human skin fibroblasts exposed to UVA light used in clinical PUVA treatment.

Human skin fibroblasts were irradiated with a clinically used UVA light source. The doses (1.1 and 3 J/cm2) were similar to those reaching the dermis during clinical PUVA treatment of psoriasis. DNA strand breaks, as determined by alkaline elution, were formed in a dose-dependent way and disappeared within 1 hr of postincubation at 37 degrees C. These findings have clinical implications since UVA-induced DNA damage may be accompanied by mutagenic and tumor promoting effects.

Studies of DNA and chromosome damage in skin fibroblasts and blood lymphocytes from psoriasis patients treated with 8-methoxypsoralen and UVA irradiation.

Exposure of human lymphocytes and skin fibroblasts in vitro to a single, clinically used dose of PUVA, i.e., 0.1 micrograms/ml of 8-methoxypsoralen (8-MOP) plus 0.9-4 J/cm2 of longwave ultraviolet radiation (UVA), lead to the formation of DNA damage as determined by alkaline elution, and to chromosome aberrations and sister chromatid exchanges (SCE). When lymphocyte-enriched plasma was obtained from psoriasis patients 2 h after oral intake of 8-MOP and then UVA irradiated (1.8-3.6 J/cm2) in vitro, an increased frequency of chromosome aberrations and SCE was observed. Normal levels of chromosome aberrations and SCE were found in lymphocytes of psoriasis patients after 3-30 weeks of PUVA treatment in vivo. A small but statistically significant increase in the SCE frequency was observed in the lymphocytes of psoriasis patients treated for 1-6 years with PUVA (mean 18.0 SCE/cell) as compared with before PUVA (mean 15.8, p less than 0.05). Skin fibroblasts of psoriasis patients analyzed 5 years after the start of PUVA treatment showed a normal number of SCE but a high fraction of filter-retained DNA in the alkaline elution assay, suggesting the presence of cross-linked DNA.

Deficient DNA repair of triple helix-directed double psoralen damage in human cells.

Damage induced by a single psoralen-modified triple helix-forming oligonucleotide has been reported to be efficiently repaired in human cells. In this study we investigated a set of psoralen coupled oligonucleotides introducing multiple lesions into the target DNA. A simian virus 40 (SV40) shuttle vector was in vitro treated with different triple helix-forming oligonucleotides and UVA radiation, leading to double psoralen adducts at the supF mutational target gene of the plasmid. After passage in the Raji human cell line the recovered vector was analysed in an indicator bacterial strain. The results show that double psoralen adducts, located at both ends of a long triple helix, cannot be repaired efficiently in human cells.

Prevalence of IgA-antiendomysium and IgA-antigliadin autoantibodies at diagnosis of insulin-dependent diabetes mellitus in Swedish children and adolescents.

OBJECTIVE: This study was conducted to investigate the prevalence of celiac disease (CD) in children and adolescents at diagnosis of insulin-dependent diabetes mellitus (IDDM) before insulin treatment was started. MATERIAL AND METHODS: At diagnosis of IDDM, and before treatment was started, 115 children and adolescents were screened for IgA- antiendomysium (EMA) and IgA-antigliadin antibodies (AGA). Those found to be EMA-positive and/or AGA-positive were investigated further with intestinal biopsy. RESULTS: Of the 115 patients, 2 had known CD at diagnosis of IDDM; of the remainder of patients, 6% (7/113) were found to be EMA-positive and 9% (10/113) were found to have AGA levels above normal. Of the 6 patients who underwent biopsy, 5 manifested villous atrophy. In addition, 2 patients with high EMA and AGA antibody titers refused biopsy, and 4 patients with low EMA and/or AGA titers were found to have normal titers at control before biopsy decision. CONCLUSION: Because the prevalence of CD at diagnosis of IDDM would seem to be 6% to 8%, screening for CD seems to be justified among patients with newly diagnosed IDDM.

Shuttle vector plasmid propagation in human peripheral blood lymphocytes facilitated by liposome-mediated transfection.

Transfection of human peripheral blood lymphocytes facilitated by a positively charged liposome preparation (Lipofectin, BRL) is 100-fold more efficient than the DEAE dextran technique for the uptake and replication of shuttle vector plasmid DNA. The yield of progeny plasmids obtained from 10 ml of blood was high enough for mutational analysis. A marked increase in the mutation frequency of the shuttle vector marker gene was noted in response to the induction of psoralen adducts in the vector. By using normal human lymphocytes, this method will permit shuttle vector analysis of DNA repair and mutagenesis in a large number of individuals. This method could also prove useful for studies of human lymphotropic viruses.

CD4+ T-lymphocytopenia without HIV infection: increased prevalence among patients with primary Sjögren's syndrome.

OBJECTIVE: Primary Sjögren's syndrome (1 degree SS) is an autoimmune disease, usually accompanied by manifest immune hyperactivity. In some cases the disease converts to malignant neoplasia. On the other hand, there are clinical similarities to HIV infection. Since the rare phenomenon of persistent depletion of CD4+ T-lymphocytes in peripheral blood without HIV infection was recently defined as idiopathic CD4+ T-lymphocytopenia (ICL), we have used the ICL criteria to investigate the prevalence of this phenomenon among 1 degree SS patients. METHODS: During the period 1988-94, 115 caucasian patients (10 males), mean age 57.8 (range 19-82) years, with 1 degree SS were prospectively studied. Lymphocyte subsets were investigated by means of monoclonal antibodies and flow cytometry. For the detection of HIV and HTLV antibodies, we used an enzyme immunoassay (for HIV-1 and HIV-2), Western blot techniques (HIV-1, HIV-2, HTLV-I and HTLV-II), and the polymerase chain reaction procedure (HIV-1, HTLV-I and HTLV-II). HIV antigens were tested for with the HIV-1 p-24 Ag test. RESULTS: Six patients with 1 degree SS fulfilled the criteria for ICL. While the clinical condition of 5 of those six patients remained stable, one patient developed malignant lymphoma three years after her disease was classified as a case of ICL. The prevalence of ICL among our 115 patients with 1 degree SS was 5.2%, which is significantly higher than the rates reported for any other patient or population group. We have estimated the relative risk of ICL in 1 degree SS patients to vary from 3.4 to 6,000 (P values of 0.0001-0.025). CONCLUSION: We suggest that subjects with ICL should be carefully examined for 1 degree SS and, if its presence is confirmed, that they should be followed with regard to the possible complications of this disease, including the development of malignant lymphoma.

Damage distribution and mutation spectrum: the case of 8-methoxypsoralen plus UVA in mammalian cells.

We determined the distribution of monoadducts and biadducts induced in the supF tRNA gene carried by the shuttle vector pZ189, after exposure to 8-methoxypsoralen (8-MOP) plus a double UVA (365 nm) irradiation. These data were compared to our previously published 8-MOP-photoinduced mutation spectrum obtained after propagation of the damaged shuttle vector in mammalian cells. One mutational hot spot in an ATAT/TATA sequence is targeted at a hot spot of biaddition. A second hot spot is not related to the presence of photoadducts either at or near the site. Moreover, it is located in a sequence which can be defined as 'mutation-prone'. Mutations occurring at GC base pairs are not targeted at sites of photoaddition, and may result from a decrease in fidelity of DNA polymerase when copying the damaged vector.

Induction of SCE by DNA cross-links in human fibroblasts exposed to 8-MOP and UVA irradiation.

To study the SCE-inducing effect of psoralen cross-links in the DNA of normal, human fibroblasts, cell cultures were exposed to PUVA (0.2-1 micrograms of 8-MOP per ml, followed by UVA irradiation at 0.04 J/cm2) and carefully washed to remove non-covalently bound psoralen. Some cell cultures were then given a second dose of UVA (1.1 J/cm2), either immediately after PUVA or 1-3 days later. By this type of treatment, cells with different proportions of DNA cross-links are obtained. The initial PUVA treatment will mainly give rise to psoralen monoadducts and only few cross-links in the DNA, and the second UVA irradiation will convert a number of the psoralen monoadducts into cross-links. SCE analysis was carried out on cells grown for 2 cell cycles in the presence of BrdUrd (10 mumoles/1). PUVA treatment alone did not induce an increase in the SCE frequency, whereas a clear increase of SCE was observed in cells treated with PUVA immediately followed by the second UVA dose. This PUVA + UVA-induced increase of SCE was also observed after incubation of the cells for 3 days at confluency, as well as when a period of 3 days at confluency was introduced between the PUVA exposure and the second irradiation with UVA. In contrast, the SCE frequency gradually returned to the normal level when PUVA + UVA-treated cells were allowed to proliferate for 1-2 days, or when a proliferation period of 2-4 days was introduced between the PUVA exposure and the second irradiation. Because the SCE frequency was not changed by the initial PUVA treatment but markedly increased by PUVA + UVA, it is concluded that psoralen cross-links are considerably more effective at inducing SCE than monoadducts. The results also indicate that SCE-inducing PUVA damage is removed very slowly if at all from the DNA of confluent cells. In contrast, repair functions that eliminate cross-links as well as monoadducts seem to become activated during cell proliferation.

Induction and repair of psoralen cross-links in DNA of normal human and xeroderma pigmentosum fibroblasts.

Skin fibroblasts from normal human subjects were exposed in vitro to long-wave ultraviolet radiation (UVA, 320-400 nm) alone, or in combination with 8-methoxypsoralen (8-MOP). DNA damage was analysed with the alkaline elution technique before and after post-treatment incubation of the cells at 37 degrees C for various times. Cells treated with UVA at 1.1 J/cm/ showed an increased DNA elution rate, which returned to the normal level within 30 min of post-treatment incubation. In cells treated with PUVA (8-MOP at 20 microgram/ml plus UVA at 0.04 J/cm2), the alkaline elution rate was not different from untreated control cells, either before or after post-treatment incubation for time up to 7 days. When the PUVA treatment was followed first by a washing, to remove any unbound 8-MOP, and then by UVA (PUVA + UVA) at 1.1 J/cm2, the alkaline elution rate decreased below the control level. During the post-treatment incubation of the PUVA + UVA-treated cells there was a gradual increase of the alkaline elution rate to a level significantly above that in control cells. This increase was observed after 30 min. It reached a maximum after 24 h and remained after 7 days of post-treatment incubation. Cells from a patient with xeroderma pigmentosum of complementation group A, which were given the same PUVA + UVA treatment, did not show any change in the alkaline elution rate during the post-treatment incubation. If, as seems likely, an increased alkaline elution rate indicates as increase of DNA breaks, and a decreased alkaline elution rate indicates the sealing of breaks and/or the formation of cross-links, and results would suggest the following: (1) UVA irradiation in itself is capable of inducing DNA breaks, which are rapidly sealed during post-treatment incubation; (2) PUVA treatment induces mono-adducts, some of which appear to remain in the DNA for at least 7 days of post-treatment incubation and can be activated to form DNA cross-links by a second dose of UVA; (3) DNA cross-links induced by PUVA + UVA can be recognized by a repair process that involves the formation of DNA breaks. This process is not observed in xeroderma pigmentosum cells of group A.

A reduced level of multiple mutation in a shuttle vector passaged in Sjögren's syndrome cells.

Sjögren's syndrome is a systemic disorder with unknown etiology, displaying many signs of autoimmunity. Although the basic mechanism of this disease is unknown, a defect in somatic mutagenesis of antibody genes has been suggested. Using a shuttle vector plasmid, we here show that the number of vectors with multiple base changes in a marker gene was reduced in B cell lines from two patients with Sjögren's syndrome (8% in both), as compared with values reported for cell lines from normal human donors (16-27%). This finding suggests that a reduction of the rate of somatic mutagenesis may influence the development of symptoms in Sjögren patients.

4-Quinolone antibiotics: positive genotoxic screening tests despite an apparent lack of mutation induction.

The effects of different 4-quinolone antibiotic derivatives (4-Qs) in a number of short-term tests commonly employed for the evaluation of genetic toxicity were studied. Incorporation of [3H]thymidine into mitogen-stimulated peripheral blood lymphocytes was strongly enhanced at a low concentration (1.56 micrograms/ml) for most of the tested 4-Qs, whereas DNA strand breakage in lymphoblastoid cells was evident only for ciprofloxacin (10 micrograms/ml and upwards), ofloxacin (80 micrograms/ml) and norfloxacin (160 micrograms/ml). Ciprofloxacin induced a significant amount of unscheduled DNA synthesis, but was found to be negative in a shuttle vector plasmid mutation test. Ciprofloxacin (80 micrograms/ml) did not inhibit enzymes involved in the early steps of pyrimidine biosynthesis. Cell growth was slightly depressed at a concentration of 20 micrograms/ml, becoming marked at 80 micrograms/ml. In conclusion, this study seeks to contribute to an improved evaluation of genotoxic screening test data, by focusing attention on the conflicting effects imposed by the 4-Qs on a battery of such tests.

Genetic aspects of psoriasis: mode of inheritance and action of PUVA on DNA.

The results of some family and experimental studies related to psoriasis are summarized. Complex segregation analysis of Lomholt's classical family material of psoriasis from the Faroe Islands gave clear evidence of a major locus (additive gene with a frequency of 0.07) plus a strong polygenic component (genetic heritability 0.87). An analysis of another family material showed complete linkage between the major locus for psoriasis and the HLA region. Treatment of cells with 8-methoxypsoralene plus a small dose of UVA induces monoadducts, some of which appear to remain in the DNA for at least 7 days of post-treatment incubation. These monoadducts can be activated to form DNA cross-links by a second, larger UVA dose. 8-Methoxypsoralene plus UVA-induced DNA cross-links can be modified by a repair process which involves the formation of DNA breaks. This process in not observed in XPA cells.