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Mazen M Sinjab,1,2 Roy S Rubinfeld,3,4 Kirsten Wagner,5,6 Edward C Parsons Jnr,7 Arthur B Cummings,8 Michael W Belin9 1Dr. Mazen Eye Clinic, Medcare Hospitals and Centres, Dubai, United Arab Emirates; 2Al Zahra Medical Group, Damascus, Syria; 3Department of Ophthalmology, Georgetown University Medical School/Washington Hospital Center, Washington, DC, USA; 4Re:Vision Private Practice, Rockville, MD, USA; 5Department of Ophthalmology, Medstar Georgetown University Hospital, Washington, DC, USA; 6Department of Ophthalmology, Medstar Washington Hospital Center, Washington, DC, USA; 7CXL Ophthalmics, Encinitas, CA, USA; 8Wellington Eye Clinic, Dublin, Ireland; 9Department of Ophthalmology, University of Arizona, Tucson, AZ, USACorrespondence: Roy S Rubinfeld 3131 Connecticut Ave, NW, #2809, Washington, DC, 20008, USAEmail Doctor@revisedeye.comPurpose: To assess the effectiveness of a novel treatment for patients with advanced corneal ectasia and loss of visual acuity (VA). Conductive keratoplasty (CK) is performed to improve VA followed by epithelium-on (epi-on) corneal crosslinking (CXL) to stabilize the cornea after CK.Methods: Retrospective, exploratory cohort study. Patients with keratoconus or postsurgical ectasia and best spectacle-corrected distance VA (CDVA) ≤ 20/40 were included. Conductive keratoplasty was performed (ViewPoint CK System, Refractec, Inc., Bloomington, MN); followed a day later by epi-on CXL (CXLUSA/CXLO, Bethesda, MD/CXLO Encinitas, CA). Measures included uncorrected distance visual acuity (UDVA) and CDVA, as well as refractive and tomographic measures and tomographic indices.Results: Data from 50 eyes of 45 patients were analyzed. Mean follow-up was 15.1 ± 12.2 months (range: 2 to 51). Overall, UDVA and CDVA improved postoperatively. Subjective refraction and tomographic metrics did not show consistent changes, but changes in tomographic indices were associated with treatment follow-up time. At the 1-year visit, mean UDVA significantly improved over baseline (P = 0.009) by approximately 3 lines; mean CDVA improved significantly (P = 10− 5) by approximately 2 lines. No eye lost lines of CDVA. Change in the Index of Surface Variance (ISV) was associated with treatment, and the D-Index trended over follow-up time.Conclusion: Conductive keratoplasty with a proprietary epi-on CXL treatment improved vision in patients with advanced ectasia This CK/epi-on CXL treatment offers the possibility of improved VA for patients with compromised vision due to ectasia.Keywords: conductive keratoplasty, CK, corneal crosslinking, crosslinking, keratoconus, ectasia, thermokeratoplasty

The p38 MAP kinase signal transduction pathway is an important regulator of proinflammatory cytokine production and inflammation. Defining the roles of the various p38 family members, specifically p38alpha and p38beta, in these processes has been difficult. Here we use a chemical genetics approach using knock-in mice in which either p38alpha or p38beta kinase has been rendered resistant to the effects of specific inhibitors along with p38beta knock-out mice to dissect the biological function of these specific kinase isoforms. Mice harboring a T106M mutation in p38alpha are resistant to pharmacological inhibition of LPS-induced TNF production and collagen antibody-induced arthritis, indicating that p38beta activity is not required for acute or chronic inflammatory responses. LPS-induced TNF production, however, is still completely sensitive to p38 inhibitors in mice with a T106M point mutation in p38beta. Similarly, p38beta knock-out mice respond normally to inflammatory stimuli. These results demonstrate conclusively that specific inhibition of the p38alpha isoform is necessary and sufficient for anti-inflammatory efficacy in vivo.

Background: Tacrolimus (FK506) is an immunosuppressive drug 50-100 times more potent than cyclosporine (CsA), the current mainstay of organ transplant rejection therapy. Despite being chemically unrelated, CsA and tacrolimus exert their immunosuppressive effects through the inhibition of calcineurin (CaN), a critical signaling molecule during T-lymphocyte activation. Although numerous clinical studies have proven the therapeutic efficacy of drugs within this class, tacrolimus and CsA also have a strikingly similar profile of unwanted side effects., Method: Our objective has been to identify a less toxic immunosuppressant through the modification of ascomycin (FK520). Quantitative in vitro immunosuppression and toxicity assays have demonstrated (see the accompanying article, p. 18) that we achieved our goal with L-732,531 (indolyl-ascomycin; indolyl-ASC), a 32-O-(1-hydroxyethylindol-5-yl) ascomycin derivative with an improved therapeutic index relative to tacrolimus., Results: We report that the attributes of indolyl-ASC may result from its distinctive biochemical properties. In contrast to tacrolimus, indolyl-ASC binds poorly to FK506 binding protein 12 (FKBP12), the major cytosolic receptor for tacrolimus and related compounds. However, the stability of the interaction between the FKBP12-indolyl-ASC complex and CaN is much greater than that of the FKBP12-tacrolimus complex. These distinguishing properties of indolyl-ASC result in the potent inhibition of CaN within T lymphocytes but may lower the accumulation of the drug at sites of toxicity., Conclusions: Indolyl-ASC may define those properties needed to increase the therapeutic efficacy of a macrolactam immunoregulant for treating both human autoimmune disease and organ transplant rejection.

Community-acquired pneumonia occurs 3 to 4 million times per year in the United States, accounting for about 500,000 hospitalizations annually. Empiric treatment is usually instituted because of a lack of early organism-specific diagnostic tests. This study compared empiric therapy with ofloxacin to standard antibiotic regimens (usually a beta-lactam with or without a macrolide) for patients hospitalized for community-acquired pneumonia. Therapy was administered to 298 patients (146 receiving ofloxacin and 152 receiving standard therapy); 227 patients (ofloxacin, 109; standard treatment, 118) were evaluable for treatment efficacy. The most common pyogenic respiratory pathogens were Haemophilus influenzae (30 isolates) and Streptococcus pneumoniae (24 isolates). There was evidence of infection with either Mycoplasma pneumoniae (38 patients), Chlamydia pneumoniae (40 patients), or a Legionella sp. (8 patients) in a total of 79 patients (35%). The clinical success rates were similar in both groups among evaluable patients (92%, ofloxacin; 87%, standard therapy) and among patients with atypical respiratory pathogens (88%, ofloxacin; 81%, standard therapy). The mean numbers (+/- the standard deviations) of intravenous doses of antibiotics were 7.5 +/- 8.0 in the ofloxacin group and 18.4 +/- 18.5 in the standard therapy group (P < 0.001); the mean number of oral doses of ofloxacin per patient was 19.7 +/- 11.2, compared with 30.2 +/- 16.0 oral antibiotic doses in the standard therapy group (P < 0.001). All treatments were well tolerated and associated with no significant clinical or laboratory abnormalities. The findings of this study indicate that ofloxacin is active against traditional bacterial pathogens as well as the major atypical respiratory pathogens. When given as monotherapy for the empiric treatment of community-acquired pneumonia, ofloxacin is as effective as standard antimicrobial therapy.

Calcineurin, a protein phosphatase found in eukaryotic cells, presents a challenging problem in heterologous protein expression because it is both heterodimeric and posttranslationally modified. In this paper, we describe the cloning of both subunits (catalytic A and regulatory B) of calcineurin from a human cDNA library and their expression at high levels in Escherichia coli. The calcineurin A subunit is expressed as an insoluble glutathione S-transferase fusion protein, while the calcineurin B subunit is soluble upon direct expression. Catalytically active holoenzyme is derived from the separately expressed subunits using a three-step refolding protocol. First, the fusion protein is solubilized, then it is cleaved at the fusion junction with thrombin, and, finally, a catalytically competent calcineurin A:calcineurin B:calmodulin complex is reconstituted by cofolding the separately purified components. In addition, we show that a similar refolding protocol can be applied to a C-terminally truncated form of calcineurin A, which lacks an autoinhibitory and calmodulin-binding domain.

The immunosuppressant FK-506 (tacrolimus) forms a complex with a ubiquitous intracellular receptor, FK-506 binding protein (FKBP12), and this complex inhibits the heterodimeric Ca2+/calmodulin-dependent phosphatase, calcineurin, an essential component of the T-cell receptor signal transduction pathway. Using a series of truncated calcineurin catalytic subunits, we show here that a region within the catalytic subunit that regulates phosphatase activity, the autoinhibitory domain, also regulates the Ca(2+)-dependent interaction of calcineurin with the FK-506.FKBP12 complex. Deletion of this domain produces constitutive activation of the phosphatase as demonstrated by transient transfection experiments in which expression of the truncated protein permitted Ca(2+)-independent induction of interleukin-2 transcription. Thus, deletion of the autoinhibitory domain is necessary and sufficient to constitutively activate calcineurin (CaN). Furthermore, CaN A467-492, an inhibitory peptide based on the autoinhibitory domain from calcineurin (ITSFEEAKGLDRINERMPPRRDAMP), inhibited dephosphorylation of the RII peptide substrate competitively with a Ki = 4 microM, consistent with binding of the autoinhibitory domain at the active site of the enzyme. To assess the role of the autoinhibitory domain in regulating the interaction of CaN with the FK-506.FKBP12 complex, we reconstituted wild type and mutant phosphatase heterodimers using in vitro transcribed and translated subunits. Association of the reconstituted calcineurin heterodimers with FKBP12 was dependent on FK-506. In the case of the wild type heterodimer, association with the FK-506.FKBP12 complex was also dependent upon Ca2+; however, mutant catalytic subunits, in which the autoinhibitory domains were deleted, associated with the drug-binding protein complex in the presence of 10 mM EGTA. These results indicate that the conserved autoinhibitory domain regulates both Ca(2+)-dependent phosphatase activity and association with the FK-506.FKBP12 complex.

The macrolide FK-506, like the cyclic undecapeptide cyclosporin A (CsA), is a potent immunosuppressant that interferes with the transcriptional activation of several early-phase genes in T lymphocytes, including that for interleukin-2 (IL-2). We compared the effects of FK-506 and CsA on transcription from the 5' upstream activating sequences (UAS) of the human IL-2 gene and several cellular and viral UAS to define cis-acting sites which may be responsive to FK-506. The UAS surveyed included the human IL-2 receptor alpha-chain, human metallothionein II, simian virus 40 early, human cytomegalovirus immediate-early, adenovirus major late, and Rous sarcoma virus long terminal repeat UAS. In addition, we studied multimers of several defined promoter elements (NFIL-2A, NF-kappa B, or NF-AT1) which are found in the UAS of the human IL-2 gene and which have been reported to be responsive to CsA when linked to a minimal promoter element (TATA box and transcription start site). Each promoter-regulatory region was fused to the bacterial chloramphenicol acetyltransferase gene and used to transiently transfect Jurkat cells. Quantitative chloramphenicol acetyltransferase assay determinations indicated that the transcriptional activity of each UAS induced upon T-cell activation was (i) completely sensitive, (ii) partially sensitive, or (iii) resistant to inhibition by CsA and FK-506. The induced transcription driven by the IL-2 promoter elements NF-AT1 and NFIL-2A could be blocked completely by FK-506 or CsA. Gel mobility shift assays indicated that the binding activities of the factors specifically interacting with these sequences were detected in activated cells regardless of whether the cells were treated with FK-506 or CsA. The results suggest that FK-506 or CsA inhibits a transacting mechanism(s) without disrupting the binding activities of these transcription factors. The degree to which each UAS was resistant to FK-506 was consistent with the level of transcription induced by phorbol myristate acetate, while UAS which were sensitive to inhibition by FK-506 were dependent on the presence of both phorbol myristate acetate and ionomycin.

Human IL-1 alpha, IL-1 beta, and TNF-alpha mRNA expression was examined in peripheral blood monocytes (PBM) from normal individuals and in primary synoviocytes isolated from patients with rheumatoid arthritis by Northern blot and in situ hybridization. Cells cultured in the presence or absence of LPS were analyzed using in vitro synthesized 35S-labeled sense or antisense RNA probes to determine the relative abundance and the cell type expressing each of the mRNA for these potent inflammatory mediators. The results indicated that 72% of the LPS-stimulated PBM expressed detectable levels of IL-1 alpha mRNA, 89% IL-1 beta mRNA, and 10% TNF-alpha transcripts. Thus, the majority of activated PBM produced both IL-1 alpha and IL-1 beta. Experiments combining immunofluorescence for IL-1 beta protein with in situ hybridization for TNF-alpha mRNA demonstrated that monocytes expressing TNF-alpha mRNA also produced IL-1 beta. Primary synoviocytes from four patients with RA were also examined for the mRNA expression of each cytokine. Northern blot analyses of total RNA isolated from 0 to 72 h after LPS- or mock-stimulation showed that IL-1 beta mRNA was the most abundantly expressed, followed by TNF-alpha. In situ hybridization revealed that IL-1 beta and TNF-alpha transcripts were detected exclusively in synovial tissue macrophages. IL-1 alpha mRNA was not detected in these cultures by either method.

The pharmacokinetics of 1-oxa-beta-lactam (LY127935), a new semisynthetic beta-lactam antibiotic, was studied in four healthy adult volunteers (mean age of 27 years, mean body surface area +/- standard error [SE] of 1.87 +/- 0.08 m(2), and mean creatinine clearance +/- SE of 116 +/- 12 ml/min per 1.73 m(2)). Immediately after completion of a 1-g, 20-min intravenous (i.v.) infusion, the mean serum level +/- SE was 70.7 +/- 8.5 mug/ml. After a 1-g intramuscular (i.m.) injection, peak serum levels occurred from 30 min to 1 h, and the mean peak serum level +/- SE was 52.3 +/- 1.6 mug/ml. Beginning at 1 h, the serum concentrations after i.m. administration were higher than those after i.v. administration. At 8 h, the mean serum level +/- SE was 3.8 +/- 0.6 mug/ml after completion of the i.v. infusion and 4.8 +/- 0.7 mug/ml after the i.m. injection. The mean serum half-lives for the beta phase i.v. and i.m. administration were similar (2.3 +/- 0.7 h and 2.4 +/- 0.2 h, respectively). The mean apparent volume of distribution +/- SE was 16.6 +/- 1.9 liters per 1.73 m(2). The mean serum clearance +/- SE of LY127935 was 85.4 +/- 12.7 ml/min per 1.73 m(2), and the mean renal clearance +/- SE was 54.5 +/- 4.4 ml/min per 1.73 m.(2) Urine concentrations of LY127935 were at least 140 mug/ml in each volunteer during the first 12 h after i.m. or i.v. administration. The mean percentages of the dose recovered in the urine +/- SE within 2 h after i.v. or i.m. administration were similar (30 +/- 4 and 34 +/- 11, respectively). Only 67 +/- 3% and 75 +/- 13% were recovered in the urine within 24 h after i.v. and i.m. administration, respectively.

Cefazolin (CZ), cephalothin (CF), cefoxitin (CX), and cefamandole (CM) were evaluated in therapy of Staphylococcus aureus infection produced in perforated table tennis balls placed intraperitoneally in rabbits. Four weeks after placement of two balls in each rabbit, a beta-lactamase producing strain of S. aureus was injected into one of the balls. Twenty-four hours later therapy was initiated with 40 mg of CZ or 80 mg of CF, CX, or CM per kg intramuscularly every 6 h. After 24 h of treatment, the mean log(10) colony-forming units per ml were 7.1 for CZ, 6.7 for CF, 6.5 for CX, and 7.2 for CM. After 72 h the mean log(10) colony-forming units per ml were 5.0 for CZ, 4.1 for CF, 3.6 for CX, and 5.6 for CM. After 8 days, the titers were 1.6/ml for CZ, 1.0 for CF, 1.9 for CX, and 3.6 for CM. CZ serum levels were about double CF and CX levels and about two-thirds of CM levels. In sterile ball fluid CZ and CM levels were more than double CF or CX concentrations. Concentrations of all four antibiotics were lower in infected balls.