This study aimed to investigate the clinical feasibility of treating severe open Lisfranc injuries by means of one-stage internal fixation with k-wires associated with vacuum sealing drainage (VSD).
The clinical outcomes of 20 cases of severe open Lisfranc joint fracture-dislocation treated by using one-stage internal fixation with k-wires associated with VSD, after debridement and suturing during emergency treatment, were reviewed.
At 6 and 12 months after surgery, the American Orthopaedic Foot and Ankle Society midfoot scores were 69.2 and 78.2, the positive rates were 75 and 85 %, and the average visual analogue scale scores were 4.3 and 1.3, respectively. The average time of internal fixation surgery was 47 min (30–70 min). There were three cases of wound-edge necrosis; however, there were no cases of skin necrosis around the incision, or deep infection. The mean time of first hospital stay was 16.1 days (10–23 days).
Treatment of severe open Lisfranc fracture and dislocation through one-stage internal fixation with k-wires in association with VSD led to fast anatomical reduction, stabilized bony structure, fast soft tissue recovery, and good short-term follow-up results.
Lisfranc joints injury; Openness; k-wire internal fixation; VSD
Women urgently need a self-initiated, multipurpose prevention technology (MPT) that simultaneously reduces their risk of acquiring HIV-1, HSV-2, and HPV (latter two associated with increased risk of HIV-1 acquisition) and prevents unintended pregnancy. Here, we describe a novel core-matrix intravaginal ring (IVR), the MZCL IVR, which effectively delivered the MZC combination microbicide and a contraceptive. The MZCL IVR contains four active pharmaceutical ingredients (APIs): MIV-150 (targets HIV), zinc acetate (ZA; targets HIV and HSV-2), carrageenan (CG; targets HPV and HSV-2), and levonorgestrel (LNG; targets unintended pregnancy). The elastomeric IVR body (matrix) was produced by hot melt extrusion of the non-water swellable elastomer, ethylene vinyl acetate (EVA-28), containing the hydrophobic small molecules, MIV-150 and LNG. The solid hydrophilic core, embedded within the IVR by compression, contained the small molecule ZA and the macromolecule CG. Hydrated ZA/CG from the core was released by diffusion via a pore on the IVR while the MIV-150/LNG diffused from the matrix continuously for 94 days (d) in vitro and up to 28d (study period) in macaques. The APIs released in vitro and in vivo were active against HIV-1ADA-M, HSV-2, and HPV16 PsV in cell-based assays. Serum LNG was at levels associated with local contraceptive effects. The results demonstrate proof-of-concept of a novel core-matrix IVR for sustained and simultaneous delivery of diverse molecules for the prevention of HIV, HSV-2 and HPV acquisition, as well as unintended pregnancy.
core-matrix design; intravaginal ring; multipurpose prevention technology; microbicides; contraception; efficacy
Women need multipurpose prevention products (MPTs) that protect against sexually transmitted infections (STIs) and provide contraception. The Population Council has developed a prototype intravaginal ring (IVR) releasing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (M), zinc acetate (ZA), carrageenan (CG) and levonorgestrel (LNG) (MZCL IVR) to protect against HIV, HSV-2, HPV and unintended pregnancy. Our objective was to evaluate the anti-SHIV-RT activity of MZCL IVR in genital mucosa. First, macaque vaginal tissues were challenged with SHIV-RT in the presence of (i) MIV-150 ± LNG or (ii) vaginal fluids (VF); available from studies completed earlier) collected at various time points post insertion of MZCL and MZC IVRs. Then, (iii) MZCL IVRs (vs. LNG IVRs) were inserted in non-Depo Provera-treated macaques for 24h and VF, genital biopsies, and blood were collected and tissues were challenged with SHIV-RT. Infection was monitored with one step SIV gag qRT-PCR or p27 ELISA. MIV-150 (LCMS/MS, RIA), LNG (RIA) and CG (ELISA) were measured in different compartments. Log-normal generalized mixed linear models were used for analysis. LNG did not affect the anti-SHIV-RT activity of MIV-150 in vitro. MIV-150 in VF from MZC/MZCL IVR-treated macaques inhibited SHIV-RT in vaginal mucosa in a dose-dependent manner (p<0.05). MIV-150 in vaginal tissue from MZCL IVR-treated animals inhibited ex vivo infection relative to baseline (96%; p<0.0001) and post LNG IVR group (90%, p<0.001). No MIV-150 dose-dependent protection was observed, likely because of high MIV-150 concentrations in all vaginal tissue samples. In cervical tissue, MIV-150 inhibited infection vs. baseline (99%; p<0.05). No cervical tissue was available for MIV-150 measurement. Exposure to LNG IVR did not change tissue infection level. These observations support further development of MZCL IVR as a multipurpose prevention technology to improve women’s sexual and reproductive health.
Evidences suggest that tumor microenvironment may play an important role in cancer drug resistance. Sphingosine kinase 2 (SphK2) is proposed to be the key regulator of sphingolipid signaling. This study is aimed to investigate whether the combination of molecular targeting therapy using a specific inhibitor of SphK2 (ABC294640), with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can enhance the apoptosis of non-small cell lung cancer (NSCLC) cells. Our results revealed that NSCLC cells' sensitivity to TRAIL is correlated with the level of SphK2. Compared with TRAIL alone, the combination therapy enhanced the apoptosis induced by TRAIL, and knockdown of SphK2 by siRNA presented a similar effect. Combination therapy with ABC294640 increased the activity of caspase-3/8 and up-regulated the expression of death receptors (DR). Additional investigations revealed that translocation of DR4/5 to the cell membrane surface was promoted by adding ABC294640. However, expression of anti-apoptosis proteins such as Bcl-2 and IAPs was not significantly modified by this SphK2 inhibitor. Overall, this work demonstrates that SphK2 may contribute to the apoptosis resistance in NSCLC, thus indicating a new therapeutic target for resistant NSCLC cells.
death receptor; NSCLC; resistance; sphingosine kinase 2; TRAIL
BACKGROUND AND AIMS: Primary gastric B-cell lymphoma is the second most common malignancy of the stomach. There are many controversial issues about its diagnosis, treatment and clinical management. “Double-hit” and “double-protein” involving gene rearrangement and protein expression of c-Myc and bcl2/bcl6 are the most used terms to describe DLBCL poor prognostic factors in recent years. However, very little is known about the role of these prognostic factors in primary gastric B-cell lymphomas. This study aims to obtain a molecular pathology prognostic model of gastric B-cell lymphoma for clinical stratified management by evaluating how the “double-hit” and “double-protein” in tumor cells as well as microenvironmental reaction of tumor stromal tissue affect clinical outcome in primary gastric B-cell lymphomas.
METHODS: Data and tissues of 188 cases diagnosed with gastric B-cell lymphomas were used in this study. Tumor tissue microarray (TMA) of formalin fixed and paraffin embedded (FFPE) tissues was constructed for fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analysis with a serial of biomarkers containing MYC, BCL2, BCL6, CD31, SPARC, CD10, MUM1 and Ki-67. Modeled period analysis was used to estimate 3-year and 5-year overall survival (OS) and disease-free survival (DFS) distributions.
RESULTS: There was no definite “double-hit” case though the gene rearrangement of c-Myc (5.9%), bcl2 (0.1%) and bcl6 (7.4%) was found in gastric B-cell lymphomas. The gene amplification or copy gains of c-Myc (10.1%), bcl-2 (17.0%) and bcl-6 (0.9%) were present in these lymphomas. There were 12 cases of the lymphomas with the “double-protein” expression of MYC and BCL2/BCL6. All patients with “double-protein” gastric B-cell lymphomas had poor outcome compared with those without. More importantly, “MYC-BCL2-BCL6” negative group of gastric B-cell lymphoma patients had favorable clinical outcome regardless clinical stage, pathological types and therapeutic modalities. And the similar better prognosis was found in the cases with low microvessel density (MVD) in tumor tissue and high expression of SPARC (SPARC≥5%) in stromal cells.
CONCLUSIONS: “Double-hit” lymphoma was rare among primary gastric lymphoma, while patients with multiple gene amplification and/or copy gains of c-Myc, bcl2 and bcl6, and “double-protein” gastric B-cell lymphomas had a poor clinical outcome. In addition, patients with MYC, BCL2 and BCL6 expression negative or low MVD in tumor tissue with high expression of SPARC in stromal cells could have better prognosis than other gastric B-cell lymphomas regardless of their clinical stage and pathological types. These results would be of very importance for clinical stratified management and precision medicine of gastric B-cell lymphomas.
Primary gastric B-cell lymphoma; Double-hit; Double-protein; MVD; SPARC; Fluorescent in situ hybridization; Immunohistochemistry and Tumor tissue microarray.
Lung fibrosis may be associated with Type-2 polarized inflammation. Herein, we aim to investigate whether radiation can initiate a Type-2 immune response and contribute to the progression of pulmonary fibrosis in tumor-bearing animals. We developed a tumor-bearing mouse model with Lewis lung cancer to receive either radiation therapy alone or radiation combined with Th1 immunomodulator unmethylated cytosine-phosphorothioate-guanine containing oligodeoxynucleotide (CpG-ODN). The Type-2 immune phenotype in tumors and the histological grade of lung fibrosis were evaluated in mice sacrificed three weeks after irradiation. Mouse lung tissues were analyzed for hydroxyproline and the expression of Type-1/Type-2 key transcription factors (T-bet/GATA-3). The concentration of Type-1/Type-2 cytokines in serum was measured by cytometric bead array. Lung fibrosis was observed to be more serious in tumor-bearing mice than in normal mice post-irradiation. The fibrosis score in irradiated tumor-bearing mice on Day 21 was 4.33 ± 0.82, which was higher than that of normal mice (2.00 ± 0.63; P < 0.05). Hydroxyproline and GATA-3 expression were increased in the lung tissues of tumor-bearing mice following irradiation. CpG-ODN attenuated fibrosis by markedly decreasing GATA-3 expression. Serum IL-13 and IL-5 were elevated, whereas INF-γ and IL-12 expression were decreased in irradiated tumor-bearing mice. These changes were reversed after CpG-ODN treatment. Thus, Type-2 immunity in tumors appeared to affect the outcome of radiation damage and might be of interest for future studies on developing approaches in which Type-1–related immunotherapy and radiotherapy are used in combination.
tumor-bearing; Type-1/Type-2; immune imbalance; radiation-induced lung injury; fibrosis
Evaluation of cancer genomes in global context is of great interest in light of changing ethnic distribution of the world population. We focused our study on men of African ancestry because of their disproportionately higher rate of prostate cancer (CaP) incidence and mortality. We present a systematic whole genome analyses, revealing alterations that differentiate African American (AA) and Caucasian American (CA) CaP genomes. We discovered a recurrent deletion on chromosome 3q13.31 centering on the LSAMP locus that was prevalent in tumors from AA men (cumulative analyses of 435 patients: whole genome sequence, 14; FISH evaluations, 101; and SNP array, 320 patients). Notably, carriers of this deletion experienced more rapid disease progression. In contrast, PTEN and ERG common driver alterations in CaP were significantly lower in AA prostate tumors compared to prostate tumors from CA. Moreover, the frequency of inter-chromosomal rearrangements was significantly higher in AA than CA tumors. These findings reveal differentially distributed somatic mutations in CaP across ancestral groups, which have implications for precision medicine strategies.
•Distinct genomic alterations were defined in prostate cancers of African American (AA) and Caucasian American (CA) men.•A novel prevalent deletion of the LSAMP locus associated with prostate cancer recurrence in AA prostate cancers•Focused evaluations of cancer genomes in the context of ethnicity have implications in enhancing precision medicine.
Men of African ancestry experience a disproportionately higher rate of prostate cancer (CaP) incidence and mortality. However, most prostate cancer genome evaluations thus far have focused on men of European ancestry. This report highlights a new recurrent genomic alteration of LSAMP locus in African American prostate cancers. Further, significant differences of two established prostate cancer driver genes, ERG and PTEN, were affirmed. This study conveys the need for careful evaluations of cancer genomes in global context with important implications for precision medicine strategies.
African American; Prostate cancer; Genome; LSAMP; ERG; PTEN
With the exploitation of rare earth ore, more and more REEs came into groundwater. This was a waste of resources and could be harmful to the organisms. This study aimed to find an efficient adsorption material to mitigate the above issue. Through doping sodium alginate (SA) with poly-γ-glutamate (PGA), an immobilized gel particle material was produced. The composite exhibited excellent capacity for adsorbing rare earth elements (REEs). The amount of La3+ adsorbed on the SA-PGA gel particles reached approximately 163.93 mg/g compared to the 81.97 mg/g adsorbed on SA alone. The factors that potentially affected the adsorption efficiency of the SA-PGA composite, including the initial concentration of REEs, the adsorbent dosage, and the pH of the solution, were investigated. 15 types of REEs in single and mixed aqueous solutions were used to explore the selective adsorption of REEs on gel particles. Scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy analyses of the SA and SA-PGA gel beads suggested that the carboxyl groups in the composite might play a key role in the adsorption process and the morphology of SA-PGA changed from the compact structure of SA to a porous structure after doping PGA. The kinetics and thermodynamics of the adsorption of REEs were well fit with the pseudo-second-order equation and the Langmuir adsorption isotherm model, respectively. It appears that SA-PGA is useful for recycling REEs from wastewater.
A sensitive and specific method for the determination of 17α-hydroxyprogesterone caproate (17-OHPC) in human plasma using high performance liquid chromatography and mass spectrometry has been developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Oasis HLB extraction cartridge prior to chromatography. Medroxyprogesterone acetate (MPA) was used as the internal standard. Chromatography was performed using Waters C18 Symmetry analytical column, 3.5 μm, 2.1 × 10 mm, using a gradient elusion with a mobile phase consisting of acetonitrile [A] and 5% acetonitrile in water [B], with 0.1% formic acid being added to both [A] and [B], at a flow rate 0.2 ml/min. The retention times of 17-OHPC and MPA were 8.1 and 5.0 min, respectively, with a total run time of 15 min. Analysis was performed on Thermo Electron Finnigan TSQ Quantum Ultra mass spectrometer in a selected reaction monitoring (SRM), positive mode using electron spray ionization (ESI) as an interface. Positive ions were measured using extracted ion chromatogram mode. The extracted ions following SRM transitions monitored were m/z 429.2→313.13 and 429.2→271.1, for 17-OHPC and m/z 385.1→276 for MPA. The extraction recoveries at concentrations of 5, 10 and 50 ng/ml were 97.1, 92.6 and 88.7%, respectively. The assay was linear over the range 0.5 - 50 ng/ml for 17-OHPC. The analysis of standard samples for 17-OHPC 0.5, 1, 2.5, 5, 10, 25 and 50 ng/ml demonstrated a relative standard deviation of 16.7, 12.4, 13.7, 1.4, 5.2, 3.7 and 5.3%, respectively (n=6). This method is simple, adaptable to routine application, and allows easy and accurate measurement of 17-OHPC in human plasma.
17-OHPC; MPA; liquid chromatography-mass spectrometry; Pregnancy
A sensitive and specific assay method for the simultaneous quantitation of 17α-hydroxyprogesterone caproate (17-OHPC), 17α-hydroxyprogesterone (17-OHP), and progesterone (P) in human plasma using high performance liquid chromatography and tandem mass spectrometry (LC-MS/MS) was developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Oasis HLB extraction cartridge prior to chromatography. Medroxyprogestrone acetate (MPA) was used as the internal standard. The compounds were separated using Waters C18 Symmetry analytical column (3.5 μm, 2.1 × 50 mm) using a gradient elusion with a mobile phase consisting of 5% methanol in water [A] and methanol [B], with 0.01% ammonium hydroxide being added to both [A] and [B], at a flow rate 0.3 ml/min. The retention times for 17-OHPC, 17-OHP, P and MPA were 4.5, 1.5, 2.5 and 2.2 min respectively, with a total run time of 7 min. The analytes were detected Micromass Quattro Micro triple quadrupole mass spectrometer in positive electron spray ionization (ESI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/z 429.10→313.10 for 17-OHPC, m/z 331.17→97.00 for 17-OHP, m/z 315.15→109.00 for P and m/z 387.15→327.25 for MPA (IS). The assay was linear over the range 1 – 200 ng/ml for 17-OHPC and 17-OHP, and 2 – 400 ng/ml for P, when 0.4 ml of plasma was used in the extraction. The overall intra- and inter-day assay variation was <15%. No significant variation in the concentration of 17-OHPC, 17-OHP or P was observed with different sample processing and / or storage conditions. This method is simple, allows easy accurate and reproducible measurement of 17-OHPC, 17-OHP and P simultaneously in human plasma, and is used to evaluate the pharmacokinetics of 17-OHPC in pregnant subjects.
17-OHPC; 17-OHP; P; MPA; liquid chromatography-mass spectrometry; Pregnancy
Prevalent infection with human herpes simplex 2 (HSV-2) or human papillomavirus (HPV) is associated with increased human immunodeficiency virus (HIV) acquisition. Microbicides that target HIV as well as these sexually transmitted infections (STIs) may more effectively limit HIV incidence. Previously, we showed that a microbicide gel (MZC) containing MIV-150, zinc acetate (ZA) and carrageenan (CG) protected macaques against simian-human immunodeficiency virus (SHIV-RT) infection and that a ZC gel protected mice against HSV-2 infection. Here we evaluated a modified MZC gel (containing different buffers, co-solvents, and preservatives suitable for clinical testing) against both vaginal and rectal challenge of animals with SHIV-RT, HSV-2 or HPV. MZC was stable and safe in vitro (cell viability and monolayer integrity) and in vivo (histology). MZC protected macaques against vaginal (p<0.0001) SHIV-RT infection when applied up to 8 hours (h) prior to challenge. When used close to the time of challenge, MZC prevented rectal SHIV-RT infection of macaques similar to the CG control. MZC significantly reduced vaginal (p<0.0001) and anorectal (p = 0.0187) infection of mice when 106 pfu HSV-2 were applied immediately after vaginal challenge and also when 5×103 pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×106 HPV16 pseudovirus particles (HPV16 PsV) was significant for MZC applied up to 24 h before and 2 h after vaginal challenge (p<0.0001) and also if applied 2 h before or after anorectal challenge (p<0.0006). MZC provides a durable window of protection against vaginal infection with these three viruses and, against HSV-2 and HPV making it an excellent candidate microbicide for clinical use.
After the surface silylation with 3-methacryloxypropyltrimethoxysilane, silica nanoparticles were further modified by 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO). The immobilization of DOPO on silica nanoparticles was confirmed by Fourier transform infrared spectroscopy, UV–visible spectroscopy, magic angle spinning nuclear magnetic resonance, and thermogravimetric analysis. By incorporating the DOPO-immobilized silica nanoparticles (5 wt%) into polypropylene matrix, the thermal oxidative stability exhibited an improvement of 62 °C for the half weight loss temperature, while that was only 26 °C increment with incorporation of virgin silica nanoparticles (5 wt%). Apparent activation energies of the polymer nanocomposites were estimated via Flynn–Wall–Ozawa method. It was found that the incorporation of DOPO-immobilized silica nanoparticles improved activation energies of the degradation reaction. Based on the results, it was speculated that DOPO-immobilized silica nanoparticles could inhibit the degradation of polypropylene and catalyze the formation of carbonaceous char on the surface. Thus, thermal stability was significantly improved.
Polypropylene; DOPO; Silica; Nanocomposites; Kinetic
A new flame retardant polycarbonate/magnesium oxide (PC/MgO) nanocomposite, with high flame retardancy was developed by melt compounding. The effect of MgO to the flame retardancy, thermal property, and thermal degradation kinetics were investigated. Limited oxygen index (LOI) test revealed that a little amount of MgO (2 wt %) led to significant enhancement (LOI = 36.8) in flame retardancy. Thermogravimetric analysis results demonstrated that the onset temperature of degradation and temperature of maximum degradation rate decreased in both air and N2 atmosphere. Apparent activation energy was estimated via Flynn–Wall–Ozawa method. Three steps in the thermal degradation kinetics were observed after incorporation of MgO into the matrix and the additive raised activation energies of the composite in the full range except the initial stage. It was interpreted that the flame retardancy of PC was influenced by MgO through the following two aspects: on the one hand, MgO catalyzed the thermal-oxidative degradation and accelerated a thermal protection/mass loss barrier at burning surface; on the other hand, the filler decreased activation energies in the initial step and improved thermal stability in the final period.
polycarbonates; flame retardance; thermal properties; activation energy
When microbicides used for HIV prevention contain antiretroviral drugs, there is concern for the potential emergence of drug-resistant HIV following use in infected individuals who are either unaware of their HIV infection status or who are aware but still choose to use the microbicide. Resistant virus could ultimately impact their responsiveness to treatment and/or result in subsequent transmission of drug-resistant virus. We tested whether drug resistance mutations (DRMs) would emerge in macaques infected with simian immunodeficiency virus expressing HIV reverse transcriptase (SHIV-RT) after sustained exposure to the potent non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 delivered via an intravaginal ring (IVR). We first treated 4 SHIV-RT-infected animals with daily intramuscular injections of MIV-150 over two 21 day (d) intervals separated by a 7 d drug hiatus. In all 4 animals, NNRTI DRMs (single and combinations) were detected within 14 d and expanded in proportion and diversity with time. Knowing that we could detect in vivo emergence of NNRTI DRMs in response to MIV-150, we then tested whether a high-dose MIV-150 IVR (loaded with >10 times the amount being used in a combination microbicide IVR in development) would select for resistance in 6 infected animals, modeling use of this prevention method by an HIV-infected woman. We previously demonstrated that this MIV-150 IVR provides significant protection against vaginal SHIV-RT challenge. Wearing the MIV-150 IVR for 56 d led to only 2 single DRMs in 2 of 6 animals (430 RT sequences analyzed total, 0.46%) from plasma and lymph nodes despite MIV-150 persisting in the plasma, vaginal fluids, and genital tissues. Only wild type virus sequences were detected in the genital tissues. These findings indicate a low probability for the emergence of DRMs after topical MIV-150 exposure and support the advancement of MIV-150-containing microbicides.
Recurrence interval of large earthquake on an active fault zone is an important parameter in assessing seismic hazard. The 2008 Wenchuan earthquake (Mw 7.9) occurred on the central Longmen Shan fault zone and ruptured the Yingxiu-Beichuan fault (YBF) and the Guanxian-Jiangyou fault (GJF). However, there is a considerable discrepancy among recurrence intervals of large earthquake in preseismic and postseismic estimates based on slip rate and paleoseismologic results. Post-seismic trenches showed that the central Longmen Shan fault zone probably undertakes an event similar to the 2008 quake, suggesting a characteristic earthquake model. In this paper, we use the published seismogenic model of the 2008 earthquake based on Global Positioning System (GPS) and Interferometric Synthetic Aperture Radar (InSAR) data and construct a characteristic seismic moment accumulation/release model to estimate recurrence interval of large earthquakes on the central Longmen Shan fault zone. Our results show that the seismogenic zone accommodates a moment rate of (2.7 ± 0.3) × 1017 N m/yr, and a recurrence interval of 3900 ± 400 yrs is necessary for accumulation of strain energy equivalent to the 2008 earthquake. This study provides a preferred interval estimation of large earthquakes for seismic hazard analysis in the Longmen Shan region.
A sensitive and specific method for the determination of cidofovir (CDV) in human plasma using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Varian® SAX extraction cartridges prior to chromatography. The internal standard was 13C5-Folic acid (13C5-FA). Chromatography was performed using a Luna C8(2) analytical column, 5 μm, 150 mm × 3.0 mm, using an isocratic elution with a mobile phase consisting of 43% methanol in water containing 12 mM ammonium acetate, at a flow rate of 0.3 mL/min. The retention times of CDV and 13C5-FA were 2.1 min and 1.9 min, respectively, with a total run time of 5 min. The analytes were detected by a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electron spray ionization (ESI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/z 280.0 → 262.1 for CDV and m/z 447.0 → 294.8 for 13C5-FA (IS). The assay was linear over the range 20 - 1000 ng/mL. Accuracy (101.6 - 105.7%), intra-assay precision (4.1 - 5.4%), and inter-assay precision (5.6 - 6.8%) were within FDA limits. No significant variation in the concentration of CDV was observed with different sample storage conditions. This method is simple, adaptable to routine application, and allows easy and accurate measurement of CDV in human plasma.
cidofovir; BK virus; liquid chromatography–mass spectrometry
Previously we showed that repeated vaginal application of a MIV-150/zinc acetate carrageenan (MIV-150/ZA/CG) gel and a zinc acetate carrageenan (ZA/CG) gel significantly protected macaques from vaginal simian human immunodeficiency virus reverse transcriptase (SHIV-RT) infection. Gels were applied either daily for 2 weeks or every other day for 4 weeks, and the animals were challenged 4–24 h later. Herein, we examined the effects of a single vaginal dose administered either before or after virus challenge. Encouraged by the vaginal protection seen with MIV-150/ZA/CG, we also tested it rectally. Vaginal applications of MIV-150/ZA/CG, ZA/CG, and CG gel were performed once 8–24 h before, 1 h after, or 24 h before and 1 h after vaginal challenge. Rectal applications of MIV-150/ZA/CG and CG gel were performed once 8 or 24 h before rectal challenge. While vaginal pre-challenge and pre/post-challenge application of MIV-150/ZA/CG gel offered significant protection (88%, p<0.002), post-challenge application alone did not significantly protect. ZA/CG gel reduced infection prechallenge, but not significantly, and the effect was completely lost post-challenge. Rectal application of MIV-150/ZA/CG gel afforded limited protection against rectal challenge when applied 8–24 h before challenge. Thus, MIV-150/ZA/CG gel is a highly effective vaginal microbicide that demonstrates 24 h of protection from vaginal infection and may demonstrate efficacy against rectal infection when given close to the time of HIV exposure.
Our laboratory has developed more than a hundred mouse monoclonal antibodies (MAbs) against Burkholderia pseudomallei and Burkholderia mallei. These antibodies have been categorized into different groups based on their specificities and the biochemical natures of their target antigens. The current study first examined the bactericidal activities of a number of these MAbs by an in vitro opsonic assay. Then, the in vivo protective efficacy of selected MAbs was evaluated using BALB/c mice challenged intranasally with a lethal dose of the bacteria. The opsonic assay using dimethyl sulfoxide-treated human HL-60 cells as phagocytes revealed that 19 out of 47 tested MAbs (40%) have prominent bactericidal activities against B. pseudomallei and/or B. mallei. Interestingly, all MAbs with strong opsonic activities are those with specificity against either the capsular polysaccharides (PS) or the lipopolysaccharides (LPS) of the bacteria. On the other hand, none of the MAbs reacting to bacterial proteins or glycoproteins showed prominent bactericidal activity. Further study revealed that the antigenic epitopes on either the capsular PS or LPS molecules were readily available for binding in intact bacteria, while the epitopes on proteins/glycoproteins were less accessible to the MAbs. Our in vivo study showed that four MAbs reactive to either the capsular PS or LPS were highly effective in protecting mice against lethal bacterial challenge. The result is compatible with that of our in vitro study. The MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS of the Burkholderia bacteria.
Lazaroids are a group of 21 -aminosteroids that lack steroid action but have a potent cytoprotective effect by inhibiting iron-dependent lipid peroxidation. However, there have been conflicting reports on the effectiveness and potency of the various lazaroid compounds. In this study, we compared the effectiveness of three major lazaroids on warm liver ischemia in dogs using a 2-hr hepatic vascular exclusion model. The agents were given to the animals intravenously for 30 min before ischemia. The animals were divided into 5 groups: Control (n=10), no treatment; Group F (n=6), U-74006F (10 mg/kg); Group G (n=6), U-74389G (10 mg/kg); Group A1 (n=6), U-74500A (10 mg/kg); Group A2 (n=6), U-74500A (5 mg/kg). The effect of treatment was evaluated by two-week animal survival, hepatic tissue blood flow, liver function tests, blood and tissue biochemistry, and histological analyses. Animal survival in all treated groups was significantly improved compared with the control (83–100% versus 30%). Elevation of liver enzymes after reperfusion was markedly attenuated in treated groups, except for an early significant increase in Group G. Postreperfusion hepatic tissue blood flow was much higher in all treated animals (50% of the preischemic level vs. 25% in the control). Lazaroids, particularly U-74500A at 5 mg/kg (Group A2), suppressed adenine nucleotide degradation during ischemia and enhanced the resynthe-sis of high-energy phosphates after reperfusion. Although structural abnormalities in postreperfusion liver tissues were markedly ameliorated in all treated groups, Group A2 showed significantly less neutrophil infiltration. Liver injury from warm ischemia and reperfusion was attenuated with all lazaroid compounds, of which U-74500A at 5 mg/kg exhibited the most significant protective activity.
The suppressed production of nitric oxide (NO), associated with endothelial dysfunction, is thought to be a cause of ischemia and reperfusion injury of the liver. But findings of the salutary effects of NO enhancement on such injury have been conflicting. In this study, we tested our hypothesis that NO enhancement would attenuate ischemic liver injury. For this purpose, an NO precursor, L-arginine, and a novel NO donor, FK409, were applied to a 2-hour total hepatic vascular exdusion model in dogs.
L-arginine was administered IV at a dose of 100 mg/kg twice (n = 5), while 300 mg/kg twice of FK409 was infused continuously into the portal vein (n = 5). The drugs were given to the animals for 30 and 60 minutes before and after ischemia, respectively. Nontreated animals were used as the control (n = 10). Two-week survival, systemic and hepatic hemodynamics indices, liver function tests, energy metabolism, and histopathology were analyzed.
Both treatments comparably augmented hepatic tissue blood flow, decreased liver enzyme release, and increased high-energy phosphate restoration during the reperfusion period, all of which contributed to rescuing all of the treated animals from the 2-hour total hepatic ischemia. In contrast, ischemia caused 70% mortality in the control group. Histologically, structural abnormality and neutrophil infiltration were markedly attenuated by the treatments. Systemic hypotension was observed in the animals treated with FK409, however.
Our data demonstrate that NO enhancement alleviates the liver injury caused by ischemia and reperfusion. The supplementation of L-arginine, rather than FK409, is considered more applicable to clinical use because of the absence of systemic adverse effects.
Lazaroids have been reported to attenuate preservation and reperfusion injury. In this study, we examined whether lazaroids can improve the outcome after 48-hr canine liver preservation and transplantation. Adult female beagle dogs were randomized into 4 dosage groups (5 animals each). Lazaroid U-74389G was intravenously administered at a dose of 0 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg to donors 30 min before harvesting and also to recipients 30 min before revascularization. Control animals (0 mg/kg) were given the lazaroid vehicle. The liver grafts were orthotopically transplanted after 48 hr of hypothermic preservation in UW solution. Lazaroid treatment significantly improved outcome after transplantation. Five-day animal survival increased from 0% in the control to 60% in the 6 mg/kg group, 100% in the 10 mg/kg group, and 80% in the 15 mg/kg group. Lazaroid protected the hepatocytes from damage during preservation, and enhanced energy charge and hepatic blood flow after reperfusion. Histological alterations were significantly less severe in the lazaroid-treated groups. The area of necrotic hepatocytes decreased from 43.7±17.7 in the control to 13.5±3.0 in the lazaroid 10 mg/kg group. These results indicate that lazaroid U-74389G has potential for improvement of clinical liver preservation.
Although much is known about the mucosal damage that occurs after intestinal warm ischemia and reperfusion and its recovery, little is known about the effect of cold preservation and transplantation on the mucosa. We studied the electrophysiological, biochemical, and histological changes of the intestinal mucosa after preservation for 24 hr and subsequent transplantation.
The small intestines from adult mongrel dogs were harvested. The intestines were orthotopically autotransplanted immediately (control group) or after preservation for 24 hr (preservation group). Jejunal and ileal tissues were taken before harvesting, at the end of preservation, 1 hr after reperfusion, and on postoperative days 3, 7, 14, and 28. The Ussing chamber method was used to study the electrophysiologic changes. Tissue maltase, diamine oxidase, and ornithine decarboxylase were measured. A histological analysis was also performed.
Control group grafts showed no evident deterioration in electrophysiology, biochemistry, or morphology. In contrast, preservation group grafts exhibited electrophysiological and biochemical degradation, complete denudation of the villi, and crypt injury (especially in the ileum) after reperfusion. Electrophysiologic function and the mucosa biochemical marker recovered within 3 days in the jejunum and within 7–14 days in the ileum; however, histological recovery of mucosal injury required 28 days in the jejunum and more than 28 days in the ileum.
Our study showed that despite severe destruction of mucosal integrity by prolonged preservation and transplantation, the intestinal mucosa has an enormous regenerative capacity. Our study also showed that regeneration was more pronounced in the jejunum than in the ileum.
Rat livers were preserved with the conventional use of UW solution for 30, 42, and 48 hr and compared with livers in which the vascular bed was expanded with an additional 10 to 60 ml UW/100 g liver. The extra UW, expressed as % liver weight, was entrapped during final portal infusion by tying off the supra- and infrahepatic inferior vena cava. A beneficial influence of the vascular expansion was most pronounced in the 40% group, with 10/10, 5/10, and 3/10 long-term survivors following transplantation after 30, 42, and 48 hr preservation versus 3/10 and 0/10 after 30 and 42 hr in the 0% controls. In separate experiments, surrogate indices of preservation quality following reperfusion explained this effect. The 40%—and, to a lesser extent, 20%—livers had higher and more uniformly distributed portal blood flow, better tissue oxygenation, smaller increases in postperfusion liver enzymes, higher adenine nucleotides and energy charge, and less histopathologic evidence of hemorrhage and congestion. Pressure changes in the vena cava fluid sump in additional experiments indicated that retrograde infusion of the trapped UW solution occurred in all of the 10–60% groups during the first 6 hr with stable pressures of 1.5 to 3 cm H20 thereafter. Collectively, these data suggest that the much discussed selective vulnerability of the microvasculature of stored allografts is due in part (or principally) to its selective lack of long-term exposure to the UW solution, which drains out of the open vessels but not from the parenchyma. The potential clinical exploitation of this concept is discussed.
Mucosal injury caused by ischemia and reperfusion has been well documented with the small intestine, but little is known about the colon. In the present study, the effect of warm and cold ischemia on the canine colon was studied and compared to that on the small intestine. After in situ flushing, the small intestine and the colon from six beagle dogs were removed and stored for 0.5, 1.5, and 3 hr at 37°C (warm ischemia) or for 1, 6, 12, 24, 36, and 48 hr at 4°C (cold ischemia). Electrophysiology, permeability, biochemistry, and histopathology of the specimens at each ischemic period and after reperfusion in the Ussing chamber were determined. Warm and cold ischemia induced duration-dependent suppression of electrophysiology in both organs, but the colonic mucosa retained higher activity of absorptive enterocytes and cryptic cells than the small intestine. Only the colon showed increased permeability of FITC-conjugated Dextran from the mucosal surface to the submucosal layer after prolonged ischemia. Changes in adenine nucleotides and purine catabolites were not markedly different between the organs. Histopathologic abnormalities during ischemia and after reperfusion were more serious with the small intestine than with the colon. Compared to warm ischemia, hypothermia lessened or delayed these morphofunctional derangements in both organs, which became universally worsened after reperfusion. Colonic mucosa receives morphofunctional derangements from ischemia and reperfusion, but the severity of the damage was much less severe in the colon than in the small intestine.