Analysis of IFN-kappa expression in pathologic skin conditions: downregulation in psoriasis and atopic dermatitis.

Interferon-kappa (IFN-kappa) is a type I IFN expressed by keratinocytes, monocytes and dendritic cells (DCs). In human keratinocytes, it is produced in response to double-stranded RNA (dsRNA) and other IFNs and protects from viral infections. In monocytes and DCs, IFN-kappa induces tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) and inhibits lipopolysaccharide (LPS)-induced IL-12. In this study, we evaluated IFN-kappa expression in skin lesions of patients with common immune-mediated inflammatory disorders using immunohistochemical techniques. IFN-kappa was not detectable in healthy skin but was strongly expressed in allergic contact dermatitis and lichen planus-affected skin. IFN-kappa was localized mainly in basal and suprabasal keratinocytes and in some leukocytes infiltrating the dermis. In contrast, IFN-kappa expression in psoriatic or atopic dermatitis (AD) pidermis was weak and detectable in only 2 of 5 patients examined. Consistently, cultured keratinocytes and monocytes obtained from psoriatic and AD patients expressed null or low levels of IFN-kappa in response to IFN-gamma, which strongly upregulates IFN-kappa in normal keratinocytes. IFN-kappa accumulated in keratinocyte cytoplasm and plasma membrane, and only limited amounts were released extracellularly. Soluble IFN-kappa did not influence keratinocyte proliferation or chemokine and membrane molecule expression, and only its membrane-associated form activated IFN-stimulated response element (ISRE) signaling. Given the difference in IFN-kappa expression levels in the skin disorders examined, IFN-kappa presence or deficiency might have different pathogenetic consequences depending also on other disease-specific intrinsic alterations.     

Inhibition of interleukin-17 prevents the development of arthritis in vaccinated mice challenged with Borrelia burgdorferi

We showed that Borrelia burgdorferi-vaccinated interferon gamma-deficient (IFN-gamma(0)) mice challenged with the Lyme spirochete developed a prominent chronic severe destructive osteoarthropathy. The immune response underlying the development of the severe destructive arthritis involves interleukin-17 (IL-17). Treatment of vaccinated IFN-gamma(0) mice challenged with B. burgdorferi with anti-IL-17 antibody delayed the onset of swelling of the hind paws but, more importantly, inhibited the development of arthritis. Histopathologic examination confirmed that treatment with anti-IL-17 antibody prevented the destructive arthropathy seen in vaccinated and challenged IFN-gamma(0) mice. Similar preventive results were obtained when vaccinated and challenged IFN-gamma(0) mice were treated with anti-IL-17 receptor antibody or sequentially with anti-IL-17 antibody followed by anti-IL-17 receptor antibody. By contrast, treatment of vaccinated and challenged IFN-gamma(0) mice with recombinant IL-17 (rIL-17) did not alter the development and progression of arthritis found in vaccinated and challenged IFN-gamma(0) mice without treatment with rIL-17. Therapeutic intervention may be a realistic approach to prevent arthritis, especially if IL-17 is involved in the perpetuation of chronic or intermittent arthritis.     

An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.     

Extreme insulin resistance due to anti-insulin receptor antibodies: a direct demonstration of autoantibody secretion by peripheral lymphocytes

A 59-year-old woman with systemic lupus erythematosus was found to have marked hyperglycemia, extreme insulin resistance and abnormally high plasma immunoreactive insulin. Her circulating erythrocytes displayed a dramatic decrease of 125I-labeled insulin binding. Both the whole serum and purified IgG fraction strongly inhibited the binding of radiolabeled insulin to control erythrocytes. These results suggested, although indirectly, the existence of antibodies to insulin receptors in the serum of the patient. To directly investigate this issue, we used an enzyme-linked solid-phase immunoassay which allows the detection and enumeration of lymphocytes secreting antibodies towards insulin receptors. Peroxidase-conjugated anti-human immunoglobulin is used to reveal the binding of antibodies to insulin receptor-coated dishes. We demonstrated that the patient's mononuclear cells, when briefly incubated in Petri dishes with partially purified insulin receptor, were able to secrete immunoglobulins of G class specifically directed to the antigen. Moreover, only a fraction of the whole population of anti-insulin receptor antibodies was directed towards the insulin binding region of the receptor, seemingly corresponding to the auto-antibodies detected with conventional binding-inhibition assay.     

Identification of New Natural CphA Metallo-β-Lactamases CphA4 and CphA5 in Aeromonas veronii and Aeromonas hydrophila Isolates from Municipal Sewage in Central Italy

Two new natural CphA metallo-β-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed k_cat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.     

Biochemical analysis of TEM-134, a new TEM-type extended-spectrum beta-lactamase variant produced in a Citrobacter koseri clinical isolate from an Italian hospital

OBJECTIVES: Kinetic characterization of TEM-134, a new TEM-type extended-spectrum beta-lactamase variant isolated from Citrobacter koseri during an Italian nationwide survey. TEM-134 is a natural derivative of TEM-2 with the following substitutions: E104K, R164H and G238S. METHODS: Recombinant TEM-134 was purified from Escherichia coli HB101 (pMGP-134) by three chromatographic steps (cation-exchange chromatography, gel permeation and fast chromatofocusing). Steady-state kinetic parameters (K(m) and k(cat)) were determined by measuring substrate hydrolysis under initial rate conditions using the Hanes linearization of the Michaelis-Menten equation. Modelling was carried out using the software Modeller (version 9.1). RESULTS: TEM-134 hydrolysed with variable efficiency (k(cat)/K(m) ranging from 5 x 10(3) to 8.0 x 10(5) M(-1) . s(-1)) penicillins, narrow-spectrum cephalosporins, cefepime, cefotaxime, ceftazidime and aztreonam, which appeared to be the best substrate. Molecular modelling of the enzyme indicated that the R164H substitution may result in a compromised omega loop in TEM-134 and this may be responsible for its narrower spectrum of activity. CONCLUSIONS: Kinetic data and molecular modelling suggested that R164H has a mild detrimental effect on the global activity of the enzyme.     

Palliative treatment of bone metastases with samarium-153 EDTMP at onset of pain

We evaluated the pain response and daily discomfort in patients suffering from a borderline degree of bone pain due to breast or lung cancer bone metastases, who had undergone early palliative radionuclide treatment. The results were compared with those from patients who had received standard analgesic therapy. Twenty-one patients (65.7 ± 3 years; 17 women) with metastatic bone cancer underwent samarium-153 (Sm-153) ethylene diamine tetramethylene phosphonate (EDTMP) administration (group A) and 18 patients (64.3 ± 8 years; 16 women) continued to receive standard analgesics (group B; control group). The patients kept a daily pain diary assessing both their discomfort and the pain at specific sites by means of a visual analog scale, rating from 0 (no discomfort–no pain) to 10 (worst discomfort–pain). These diaries were reviewed weekly for 2 months and three physicians rated the pain response on a scale from ?2 (considerable deterioration) to +2 (considerable improvement). Baseline characteristics were similar in both groups. The reduction of total discomfort and of bone pain in group A was significantly greater compared to group B (p < 0.0001). A significant improvement of clinical conditions was observed in group A, where the physician rate changed from ?1 to 1, compared to group B in which the rate changed from ?1 to 0. Sm-153 EDTMP therapy can be considered for patients with bone pain from breast and lung cancer in advance, i.e., before the establishment of severe pain syndrome.