Absence of rapid selection for acyclovir or penciclovir resistance following suboptimal oral prodrug therapy of HSV-infected mice.

BACKGROUND: Acyclovir (ACV) resistant herpes simplex virus (HSV) isolates can be readily selected in animal infection models receiving suboptimal ACV treatment, however no comparative studies of the emergence of resistance following suboptimal treatment with valacyclovir (VCV) or famciclovir (FCV), the prodrugs of acyclovir and penciclovir, respectively, have been reported. METHODS: Mice (n = 30) were infected with HSV type 1 or 2 in the ear pinnae and administered oral prodrugs at one fifth a dose previously shown to be effective. To select and amplify drug-resistant HSV, a total of seven consecutive in vivo passages with suboptimal treatment were performed for each virus sample and progeny virus from each passage was characterized by the plaque reduction (PRA) and plating efficiency assays (PEA). RESULTS: No drug-resistant HSV-2 and only a single drug-resistant HSV-1 variant were identified. Virus recovered from the first three sequential passages of this HSV-1 sample was susceptible by PRA, although the proportion of resistant virus recovered gradually increased upon passage. The resistant HSV-1 phenotype was confirmed by PRA after four sequential passages in mice. Unexpectedly, this in vivo-selected drug-resistant HSV-1 failed to yield an infection completely refractory to treatment in subsequent passages. CONCLUSIONS: Sub-optimal therapy of immunocompetent mice with either VCV or FCV did not readily select for HSV-mutants resistant to either ACV or PCV, suggesting that selection of resistance with either prodrug remains difficult using this system. Futhermore, this study suggests that the PEA may represent a useful adjunct to the PRA for monitoring alterations in the proportion of drug-resistant virus even when no change in IC50 is apparent.

Identification and initial characterization of 5000 expressed sequenced tags (ESTs) each from adult human normal and osteoarthritic cartilage cDNA libraries.

OBJECTIVE: To prepare, sequence and analyse adult human cartilage cDNA libraries to study the gene expression pattern between normal and osteoarthritic cartilage. METHODS: Poly A(+)RNA from adult human normal and osteoarthritic articular cartilage was isolated and used to prepare cDNA libraries. Approximately 5000 ESTs from each library were sequenced and analysed using bioinformatic tools. The expression of select genes was confirmed by Northern blot and in situ hybridization analysis. RESULTS: Multiple gene families including several classical cartilage matrix protein encoding genes were identified. Approximately 28-40% of the genes sequenced from these libraries were novel, while half of the genes encoded known proteins and 4-6% of the genes encoded novel homologs of known proteins. Several known genes, whose expression has not been reported previously in cartilage, were also identified. We have confirmed the cartilage expression of three known (CTGF, CTGF-L and clusterin) and two novel homologs of known genes (PCPE-2 and Gal-Nac transferase) by Northern blot and in situ hybridization analysis. CONCLUSION: This is the first report of the preparation and sequencing of cDNA libraries from adult human normal and osteoarthritic articular cartilage. Further analysis of genes identified from these libraries may provide molecular targets for diagnosis and/or treatment of osteoarthritis (OA).

Characterization of herpes simplex viruses selected in culture for resistance to penciclovir or acyclovir.

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv(r) clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv(r) mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.

Rapid isolation of tissue-specific genes from rat kidney.

A systematic effort to isolate kidney-specific genes was performed using recently described PCR-select methodology. Using this technique, a kidney-specific mini-gene library was generated and a number of kidney-specific genes that share significant homology to previously characterized kidney genes from rats and other species were isolated. These included three renal-specific transporters (an ADH water channel, the anion transporters RST and ROAT1), a cell adhesion molecule (K-cadherin) and a kidney-specific protein upregulated in renal carcinoma (DD96). In addition, we isolated two novel genes from a rat kidney. One of the genes shares limited homology to rat profilin-1 while the other did not share any similarity to genes in the Genbank. Northern blot analysis revealed that the mRNA for each of these genes is expressed in a highly kidney-restricted fashion. Our results suggested that tissue-specific genes can be rapidly isolated and characterized using PCR-select techniques and this methodology may be generally applicable to isolate specific genes from a variety of tissues.

Identification of a novel kidney-specific gene downregulated in acute ischemic renal failure.

To gain further insights into the molecular mechanisms involved in acute renal failure, we have isolated a new gene from rat and human, named KSP32 (kidney-specific protein with a molecular mass of 32 kDa). KSP32 encodes a novel gene that shows little homology to other mammalian proteins. It, however, shares extensive homology with several proteins found in the nematode Caenorhabditis elegans and plants. The expression of KSP32 mRNA is highly restricted to kidney. In situ hybidization analysis revealed that the expression of KSP32 mRNA was prominent in the boundary of kidney cortex and outer medulla, exhibiting a raylike formation extending from the medulla into the cortex. Finally, KSP32 mRNA was dramatically downregulated in rat following induction of acute ischemic renal failure. Rapid loss of KSP32 mRNA expression was observed beginning at approximately 5 h following renal injury and mRNA levels remained depressed for at least 96 h. Both KSP32 mRNA levels as well as renal function recovered 14 days after injury. Administration of an endothelin receptor antagonist (SB-209670), known to restore renal function, significantly increased KSP32 expression.

Comparison of a maedi-visna virus CA-TM fusion protein ELISA with other assays for detecting sheep infected with North American ovine lentivirus strains.

A maedi-visna virus CA-TM fusion protein ELISA (MVV ELISA) was evaluated for the detection of antibody in sheep infected with North American ovine lentivirus (OvLV). The results of the MVV ELISA were compared with other assays for OvLV antibody and with viral infection in an intensively studied group of 38 sheep with a high prevalence of OvLV infection and disease. The sensitivity, specificity, and concordance of assays for OvLV antibody (MVV ELISA, indirect ELISA, Western blot, and AGID), virus (virus isolation, PCR, antigen ELISA), and OvLV-induced disease in each animal were compared with OvLV infection status as defined by a positive result in two or more of the assays. Five sheep met the criteria for absence of OvLV infection. The sensitivity of the MVV ELISA in detecting OvLV infected sheep was 64%, whereas the sensitivity of the other three tests for antibody ranged from 85 to 94%. All the antibody assays were 100% specific in this group of animals. Of the assays for virus, the PCR test had the highest sensitivity and the best concordance with OvLV infection, but it also had the lowest specificity of any of the virus or antibody assays. Among the antibody tests, the concordance of the MVV ELISA compared most favorably with the AGID test for detecting OvLV-infected sheep. Analysis of serum samples from 28 lambs experimentally-infected with one of three North American strains of OvLV suggested that there were no significant strain differences detectable by antibody assay. Twenty virus-inoculated lambs were positive by both the MVV ELISA and the AGID test, five lambs were MVV ELISA negative and AGID test positive, and three lambs were MVV ELISA positive and AGID test negative. No pre-inoculation samples were positive by either assay. In a longitudinal study involving seven lambs, antibodies to OvLV were detected by AGID 3-5 weeks post-inoculation, but were not detected by MVV ELISA until 5-10 weeks post-inoculation. Among 128 naturally and experimentally-infected sheep that were seropositive in the AGID test, the overall sensitivity of the MVV ELISA was higher in the naturally infected sheep (84%) than in the experimentally infected sheep (69%). The data indicated that the MVV ELISA represents a less sensitive, but specific alternative for the detection of OvLV antibodies.

Identification, molecular cloning, expression, and characterization of a cysteinyl leukotriene receptor.

The cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory disorders, in particular asthma, for which the CysLT receptor antagonists pranlukast, zafirlukast, and montelukast, have been introduced recently as novel therapeutics. Here we report on the molecular cloning, expression, localization, and pharmacological characterization of a CysLT receptor (CysLTR), which was identified by ligand fishing of orphan seven-transmembrane-spanning, G protein-coupled receptors. This receptor, expressed in human embryonic kidney (HEK)-293 cells responded selectively to the individual CysLTs, LTC(4), LTD(4), or LTE(4), with a calcium mobilization response; the rank order potency was LTD(4) (EC(50) = 2.5 nM) > LTC(4) (EC(50) = 24 nM) > LTE(4) (EC(50) = 240 nM). Evidence was provided that LTE(4) is a partial agonist at this receptor. [(3)H]LTD(4) binding and LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor were potently inhibited by the structurally distinct CysLTR antagonists pranlukast, montelukast, zafirlukast, and pobilukast; the rank order potency was pranlukast = zafirlukast > montelukast > pobilukast. LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor was not affected by pertussis toxin, and the signal appears to be the result of the release from intracellular stores. Localization studies indicate the expression of this receptor in several tissues, including human lung, human bronchus, and human peripheral blood leukocytes. The discovery of this receptor, which has characteristics of the purported CysLT(1) receptor subtype, should assist in the elucidation of the pathophysiological roles of the CysLTs and in the identification of additional receptor subtypes.

Analysis of sheep T-cell receptor beta-chain heterogeneity.

We analyzed nucleotide and deduced amino acid sequence heterogeneity of sheep T-cell receptor beta-chain cDNAs isolated from an anchored-polymerase chain reaction library. Evaluation of 34 individual rearrangements has defined 18 new beta-chain variable region sequences which have been clustered into 13 families. Presumptive allelic polymorphisms of four of these variable regions have been defined, as well as ten distinct beta-chain joining region sequences. The present analysis indicates that sheep T-cell receptor beta-chains are composed of characteristic leader, variable, joining, and constant region sequences, and that imprecise joining and N-region addition contribute significantly to diversity in the third hypervariable region. Thus, it appears that sheep, like all other mammals studied to date, employ somatic rearrangement of multiple germline genes to create beta-chain heterogeneity. These findings have allowed us to estimate the diversity of the sheep T-cell receptor beta-chain variable region repertoire, and they provide information that will permit the evaluation of the role that specific T-cell populations play in naturally occurring and experimental diseases of sheep.

Multifaceted inhibition of anti-tumour immune mechanisms by soluble tumour necrosis factor receptor type I.

Soluble tumour necrosis factor receptor type I (sTNFRI) is a potent inhibitor of TNF with the potential to suppress a variety of effector mechanisms important in tumour immunity. That sTNFRI influences tumour survival in vivo is suggested by results from human clinical trials of Ultrapheresis, an experimental extracorporeal treatment for cancer. While the considerable clinical benefit provided by Ultrapheresis is correlated with the removal of plasma sTNFRI, there is no direct evidence that sTNFRI inhibits immune mechanisms which mediate tumour cell elimination. To evaluate formally the ability of sTNFRI to inhibit these mechanisms, we have engineered sTNFRI production into the TNF-sensitive murine fibrosarcoma cell line, L929. Soluble TNFRI-secreting L929 cells display increased resistance to direct lysis by TNF, and to lysis by syngeneic lymphokine-activated killer cells and cytotoxic T cells. These findings confirm the suggestion that sTNFRI inhibits immunological mechanisms important in tumour cell eradication, and further support a role for sTNFRI in tumour survival in vivo. In addition, these observations suggest the development of methods for more specific removal and/or inactivation of sTNFRI as promising new avenues for cancer immunotherapy.

Mechanisms of oxidant-mediated cell injury. The glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide.

Inhibition of ADP phosphorylation by both glycolysis and mitochondria in P388D1 cells exposed to H2O2 is described. Net glucose uptake and lactate production were inhibited by oxidant exposure (ED50 = 50-100 microM). Glycolysis was specifically inactivated at the glyceraldehyde-3-phosphate dehydrogenase step by three independent mechanisms: (a) direct inactivation of the intracellular enzyme (ED50 approximately equal to 100 microM); (b) reduction of the intracellular concentration and redox potential of its nicotinamide cofactors; and (c) a cytosolic pH shift further from the enzyme optima. Consistent with inhibition of glycolysis at the glyceraldehyde-3-phosphate dehydrogenase step, a rise in the intracellular concentration of glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, and fructose 1,6-bisphosphate was observed. The calculated combined inhibition of glyceraldehyde-3-phosphate dehydrogenase activity could be reasonably correlated with the depression in glycolytic flux rate with the appropriate modeling. The steady-state contribution by mitochondria to the total intracellular ATP pool was indirectly determined by the use of various metabolic inhibitors and was found to rapidly decline following exposure to 300-800 microM H2O2. The inhibition of ADP phosphorylation appeared to be related more to the direct inhibition of the ATPase-synthase complex rather than to the diminished capacity of the respiratory chain for coupled electron transport. Both the estimated rates of ADP phosphorylation by glycolysis and mitochondria and the estimated rate of ATP hydrolysis by ongoing metabolism were utilized to model the approximate decline in intracellular ATP expected at 15-min exposure to various H2O2 concentrations. Theoretical calculations and the measured intracellular ATP status were in good agreement. Oxidant exposure for 15 min resulted in dose-dependent killing of the cells (ED50 = 500 microM), indicating a close correlation between H2O2-mediated loss of intracellular ATP and cell viability. The possible contribution of impaired energy homeostasis during oxidant-mediated injury to the process of cell dysfunction and death is discussed.

Experimental pulmonary inflammatory injury in the monkey.

Inflammatory pulmonary injury was induced in Macaca mulatta rhesus monkeys by the intrabronchial instillation of the formylated peptide norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA). Indicators of pulmonary injury included an increase in mean protein content of bronchoalveolar lavage (BAL) fluid from 0.51 mg/ml in untreated animals to 3.74 mg/ml and 6.64 mg/ml in FNLP- and PMA-treated animals, respectively, the appearance of a diffuse pulmonary infiltrate in chest roentgenograms, and histologic evidence of a predominantly neutrophilic leukocytic infiltration. Concomitant with the appearance of pulmonary injury was the generation of proteases and oxidants in the BAL fluids. Neutrophil elastase, bound to alpha 1-protease inhibitor (alpha 1-PI), was found to increase from 0.47 micrograms/ml in untreated monkeys to 0.99 micrograms/ml in FNLP-treated animals and 1.23 micrograms/ml in monkeys receiving PMA. Radioiodinated human prekallikrein, instilled for 2 min into the inflammatory site and retrieved by lavaging, was found to have undergone proteolytic cleavage; this cleavage was not consistently inhibitable with the inclusion of antibody to elastase. BAL fluids were shown to contain an amidolytic activity when tested on the synthetic substrate H-D-pro-phe-arg-pNA. This activity was partially inhibitable with known inhibitors of active Hageman factor and kallikrein. beta-Glucuronidase levels in the BAL fluids increased from 0.85 U/ml to 4.36 U/ml and 8.25 U/ml in FNLP- and PMA-treated animals, respectively. Myeloperoxidase (MPO) levels also increased from 1.37 OD U/ml X min to 16.59 and 30.47 OD U/ml X min in the same groups of animals. Oxidant generation was also assessed in several different ways. The specific activity of the oxidant-sensitive inhibitor alpha 1-PI recovered in the BAL fluid decreased from 0.80 in control samples to 0.57 and 0.65 in FNLP- and PMA-treated animals. That this inactivation was due to oxidant injury of the molecule was confirmed by the return to full activity of four out of five BAL samples after their incubation with the reducing agent dithiothreitol in the presence of methionine sulfoxide peptide reductase. The specific activity of catalase in the BAL fluids of animals given 3-amino, 1,2,4 triazole (AT) 1 h before lavaging showed drops from 0.97 in untreated monkeys to 0.04 in FNLP-treated and 0.49 in PMA-treated monkeys. MPO levels also fell in the AT-treated injured animals from 16.59 to 0.85 delta OD/min X ml in FNLP animals in the absence and presence of AT, and 30.47 to 0.60 delta OD/min X ml in PMA-treated animals. Inhibition of MPO by AT was shown in vitro to be H2O2 dependent. Total glutathione levels in the BAL fluids did not change appreciably after FNLP or PMA treatment. These studies present substantial evidence of the generation of both proteases and oxidants during the establishment of acute pulmonary inflammatory injury in an experimental primate model.

The two-layer rhytidectomy.

In order to consider segmental correction of the aging face, pertinent disfigurements and underlying anatomy must be analyzed. Bony structures such as chin and malar eminence might be augmented. Submental exploration should be considered when fatty herniation or anterior platysmal banding is diagnosed. The superficial muculoaponeurotic system (SMAS)-platysmal muscle system should be undermined and plicated posteriorly when excessive laxity is noted intraoperatively. Thus, SMAS rotation flap is best for the cheek region, and imbrication may have a place in the neck. We are all witness to the classic techniques that have stood the test of time. However, more radical surgery may be indicated in certain patients. At present we feel there are few indications for complete horizontal platysmal incision and muscle flap rotation. No long-range study has been reported showing the complication rate or the endurance of this platysmal section technique. We feel that the more conservative platysmal surgery, combined with submental surgery and secondary "tuck-up" procedures as required, is the procedure of choice.

Host-range and partial characterization of several new bacteriophages for Bacillus megaterium QM b1551.

Several phages infecting Bacillus megaterium QM B1551 have been isolated from the soil and partially characterized. These phages, designated MP9 to MP50, were tested for host-range on several strains of B. megaterium and 13 other Bacillus species. All the phages only infected B. megaterium and on the basis of host-range patterns, 23 groups could be distinguished. The phage patterns also distinguished subgroups of B. megaterium strains within the species and should be useful in phage typing. The phages have varying sensitivities to heat, salts and organic solvents and are all double-stranded DNA phages. Thirty-two have been examined by electron microscopy and are Bradley types A, B and C. This is the first large collection of B. megaterium phages that has been characterized.