Mannose-Binding Lectin In The Defence Against Genital Candida Infections
MÅRDH PE, HENIC E, THIEL S
Dept of Obstetrics and Gynecology, Lund University, Sweden and Dept. of Medical Microbiology and Immunology, Aarhus University, Denmark
Background: Mannose-binding lectin (MBL) is active in the innate immune defence against genital infections. MBL binds to C. albicans and is considered to protect against vulvovaginal candidiasis (VVC) and such recurrent infections (RVVC). RVVC is not a chronic infection but characterized by repeated attacks. RVVC cases are often candida culture-negative in spite of symptoms and signs of vaginitis in between the attacks.
Material and Methods: Twenty-nine women with a history of RVVC were investigated, who had all consulted the Gyneaecological Department at the University Hospital, Lund. The comparison group consisted of 30 women who were staff at the Department. All patients reported to a standardized history form and were subjected to a speculum investigation.
Cultures of vulvar and vaginal samples collected from the lateral vaginal wall and the posterior fornix were grown on Chromagar, which allowed speciation of any candida isolate. Serum levels of MBL were determined by a sandwitch time-resolved immunofluorometric assay, using anti-MBL coated microtiter wells containing, samples, which were washed and incubated with biotinylated anti-MBL followed by europium-labelled streptavidin and measured by time-resolved flurometry.
Results: The serum levels of MBL in the RVVC group, age 21-58 (mean 31) years, ranged from <10 to 5892 ng/mL (mean 1590 ng/mL). The corresponding figures for the comparison group, age 21-64 (mean 41) years, were <10 to 2987 ng/mL (mean 920 ng/mL). The difference was significant (p=0.006). The levels were higher in culture-positive (44,8%) than culture-negative RVVC patients (p=0,02). Candida albicans made up all, but one (C. glabrata) of the isolates. RVVC who were candida-negative when consulting had MBL levels comparable to the controls. There was no difference in MBL levels between women of different age (10 years ranges).
Conclusions: MBL levels were higher in women with a history of RVVC than in the controls as well as higher in those who at sampling occasion were candida-positive compared with candida-negative women. MBL may have a therapeutic effect in cases of recurrent gentital candida infections.
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Prospective, Structured Data Collection For Recombinant FVIIa: The STER Experience On 55 Surgical Interventions In Patients With Congenital FVII Deficiency
MARIANI G, DOLCE A and INGERSLEV J for the STER and the IRF7 Study Group
Haematology, University of L’Aquila, Italy
Background: Being bleeding a major complication of surgery, its control is essential for the outcome of any operation. This is even more important when surgery is performed in patients with Congenital Bleeding Disorders (CBDs). CBDs are usually identified by a positive bleeding history, but these patients may undergo a surgical operation undiagnosed, usually because of a negative history. The severity of bleeding in the surgical scenario depends on the type of intervention, the severity of the CBD, the presence of co-morbidities and the efficacy of the replacement therapy. In FVII deficiency, a prevalence of 23.5% of peri- and post-operative bleeds was reported by us, together with not a negligible prevalence of thromboses (3.5% of patients), spontaneous or treatment-related. The occurrence of an inhibitor to the missing factor is also a severe complication of treatment. A structured evaluation of a large number and variety of surgical cases, is an excellent method to perform the pharmaco-vigilance of replacement therapies.
Methods: Within the frame of a multicentre, prospective, observational study, using an online registry, we have evaluated 55 surgical interventions (27 “major”, 28 “minor”) treated with recombinant FVIIa (n=46) or a plasma-derived FVII concentrate (n=9), to protect 48 different patients with a FVII deficiency (18 severe & moderate and 30 mild) from surgery-related bleeding.
Results: Mean daily dosages ranged from 1.2 to 87 μg/Kg/bw for rFVIIa and from 6 to 34 IU/Kg/bw for the pdFVII concentrate. Replacement therapy was carried out for a median of 9 d. (1-37) for the “major” and 4 d. (1-16) for the “minor” surgeries. A mild bleeding event occurred (2nd d ) in a patient treated with rFVIIa. An antibody to FVII (max titre 10 BU) appeared in a patient treated with rFVIIa who, nonetheless, had received plasma-derived products, previously. No thrombotic events occurred.
Conclusions: Both kind of replacement therapies proved safe and efficacious for the treatment of FVII deficiency. Major side effects, clearly related to either the pdFVII concentrate of rFVIIa, did not occur. The wide range of replacement therapy duration and dosages outlines the need for a consensus focused on the identification of optimal replacement therapy schedules in both minor or major surgeries.
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The Role OF Heat Shock Proteins IN Oral Cancer: A Possible New Target IN Antineoplastic Therapy?
MARKOPOULOS AK, DELIGIANNI E, ANTONIADES DZ
School of Dentistry, Aristotle University, Thessaloniki, Greece
Background: Heat shock proteins (HSPs) are a group of highly immunogenic proteins with an exceptional degree of conservation. They are expressed or increased in response to various biological stresses, while their best known physiological role is to act as molecular chaperones.
Methods: Immunohistochemistry staining was conducted to study HSP70 expression in 50 paraffinized tissue samples; 30 oral squamous cell carcinomas, 10 leukoplakias with dysplasia) and 10 samples from normal oral tissue.
Results: Our preliminary results showed that in squamous cell carcinoma, the cytoplasm and the cell membrane of the tumor cells was often positive for HSP70. Dysplastic lesions were positive to a lesser extent for HSP70. Samples from normal oral tissue were negative for HSP70.
Conclusions: It is concluded that HSP70 immunoexpression could be a marker for the presence of epithelial dysplasia or epithelial malignant transformation. Our results agree with certain of the literature. Overexpression of HSPs has been observed in certain cancers and their physiopathologic activity in tumorigenesis is related to cell growth and differentiation. It is known that members of the heat shock protein family (i.e. HSP70 and HSP90) are often associated with cell-cycle-related proteins including p53, Cdk4, c-myc, pRb and p27. Along with their intracellular chaperoning function, HSPs have been found to play key roles in cancer immunity by chaperoning tumor-derived peptides to major histocompatibility complex (MHC) class I molecules to elicit an anticancer immune response mediated by cytotoxic T lymphocytes. It has been demonstrated that physical as well as chemical stress increases the amount of cytoplasmic and plasma membrane-bound Hsp70, while normal cells even after exposure to stress fail to express HSP70 on their plasma membrane. Additionally, a tumor-selective cytoplasmic - membrane expression has been found to correlate with an increased sensitivity to the lytic activity mediated by natural killer cells. Taking all these into account we can postulate that HSP70 in the future can be an ideal target structure for a molecular-based anticancer immunotherapy.
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Activation Of Exploratory Behavior In Senescence Accelerated OXYS Rats By Stimulation Of Cell-Mediated Immune Response With BCG Vaccine
MARKOVA EV1, KOLOSOVA NG2, ABRAMOV VV1, KOZLOV VA1
1Inst. of Clinical Immunology, Novosibirsk, Russia; 2Inst. of Cytology and Genetics, Novosibirsk, Russia
Background: Changes in cognitive and emotional spheres are typical of aging. Their prevention is an obligatory condition providing active longevity of elderly people. We demonstrated that senescence-accelerated OXYS rats constitute appropriate model for studying ageing processes. Changes in exploratory activity and the degree of anxiety in OXYS rats are significantly related to activity of cellular immune reactions. The aim of our study was to clear out whether it was possible to modify the behavior of OXYS rats by stimulating cellular immune reactions.
Methods: This study included 40 3-month-old male OXYS and 10 Wistar rats. All animals were tested in an “open field”, after which some animals were intraperitoneally injected with dry tuberculous BCG vaccine (Allergen Firm) in doses of 50 or 600 µg/kg in 0.5 ml RPMI-1640. Controls were injected with the same volume of the solvent (RPMI-1640) under similar conditions. Two weeks after injection the parameters of exploratory behavior, cellular immune reactions were repeatedly evaluated.
Results: During the first open-field testing horizontal and vertical motor activities of OXYS rats were, as expected, markedly lower than of Wistar rats. High anxiety characteristic of OXYS rats manifested in a 3-fold lower number of grooming reactions in comparison with repeatedly tested Wistar rats and 5-fold greater number of boluses. Stimulation of the cellular component of the immune response by injection of BCG vaccine significantly modified horizontal motor activity of animals. The effect was dose-dependent and peaked after injection of 600 µg/kg vaccine: in this case horizontal motor activity increased 3-fold and did not differ from that of Wistar rats. Animals receiving the maximum dose of the vaccine did not differ from Wistar rats also by the parameters of vertical motor activity and number of grooming reactions.
Conclusions: Stimulation of cell-mediated immune response with BCG vaccine caused a dose-dependent activation of exploratory behavior in senescence accelerated OXYS rats. The possibility of correction of behavioral changes associated with early aging of OXYS rats by stimulation of cellular immune reactions is a direct proof of the involvement of neuroimmune relationships in the process of aging.
Authors’ disclosure statement: OXYS rats bred at Institute of Cytology and Genetics (Novosibirsk, Russia) is an adequate model of accelerated aging and aging-related neurodegenerative processes. OXYS rats show shortened lifespan coupled with several geriatric disorders such as early cataract, macular dystrophy, hypertension, suppression of the cell component of the immune system, as well as changes in cognitive and emotional spheres (increased anxiety, disturbances in associative learning, decreased exploratory activity in the open field test) develop in OXYS rats by the age of 3 months.
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Effective Drugs In Psychiatry: Magic Bullets Or Hand Grenades?
MARKS DM1, PATKAR AA1
1Duke Clinical Research Institute. Durham, North Carolina, USA
Background: Neuropharmacological research has improved understanding of the molecular basis of psychiatric diseases including depression and schizophrenia. As a result, drug development has focused on identifying compounds that selectively influence the relevant molecular targets. Although selective agents may have tolerability advantages, it is unclear whether increases in selectivity have enhanced or compromised efficacy in the treatment of depression and schizophrenia.
Methods: Literature was reviewed to compare relative efficacy among antidepressants and antipsychotics with particular focus on the neuropharmacological profiles of these drugs. Selective serotonin reuptake inhibitors (SSRIs) were compared to broader antidepressants affecting serotonin and norepinephrine such as serotonin/norepinephrine reuptake inhibitors (SNRIs), monoamine oxidase inhibitors, and mirtazapine. Additionally, the dopamine-specific antipsychotic haloperidol was compared to agents with broader receptor-binding characteristics including clozapine and olanzapine, and available comparative efficacy data among antipsychotics in general were evaluated with attention to the receptor selectivities of the various drugs.
Results: There are some indications of a statistically-but not necessarily clinically- significant advantage in antidepressant efficacy advantage of drugs enhancing neurotransmission of both serotonin and norepinephrine compared to SSRIs. There is no consistent evidence that SSRIs or SNRIs are superior in efficacy to older non-selective antidepressants. Newer non-selective antidepressant agents under development such as the triple uptake inhibitors hold promise. Similarly, studies support greater antipsychotic efficacy with the broad spectrum agents such as clozapine compared to haloperidol in the treatment of schizophrenia.
Conclusions: 1) Broad spectrum antidepressants and antipsychotics have demonstrated comparable and sometimes superior efficacy compared to more selective agents. 2) The advantages of selective agents over the broad spectrum agents have been in tolerability as opposed to efficacy. 3) Since multiple neurotransmitter systems are affected in most psychiatric disorders, future drug development efforts should focus on developing broad spectrum agents that optimize efficacy without compromising tolerabilty.
Authors’ disclosure statement:
Dr. Marks has received research support from Merck and National Institutes of Health and is on the speaker’s bureau of Eli Lilly and Company and Pfizer. Dr. Patkar is a consultant for Bristol-Myers Squibb, GlaxoSmithKline, and Reckitt Benckiser; is on the speaker’s bureau of Bristol-Myers Squibb, GlaxoSmithKline, and Reckitt Benckiser; has received research support from National Institutes of Health, AstraZeneca, Bristol-Myers Squibb, Forest, GlaxoSmithKline, Janssen, McNeil Consumer and Specialty Inc, Organon, Jazz Pharmaceuticals, and Pfizer.
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Dual Inhibitors Targeting Matrix Metalloproteinases And Carbonic Anhydrases As Potential Anticancer Drugs
MARQUES SM, SANTOS MA
Centro de Química Estrutural, Instituto Superior Técnico, Lisboa, Portugal
Background: Matrix metalloproteinases (MMPs) and carbonic anhydrases (CAs) are two classes of zinc-dependent enzymes with different roles and catalytic targets, such as the degradation of most of the extracellular matrix (ECM) proteins, and the regulation of the CO2/HCO3- equilibrium in the cells, respectively. Both families have isoforms which have been proved to be involved in several stages of carcinogenic processes, and so the selective inhibition of these enzymes might be of interest in cancer therapy.
Methods: We report herein the design, synthesis and in vitro evaluation of a series of compounds possessing the iminodiacetic acid as the main backbone and two functional groups attached, namely the hydroxamic and the arylsulfonamide (ArSO2NH2) moieties, to enable the inhibition of MMPs and CAs, respectively. Docking studies were also performed in order to investigate the binding interactions formed between ligand-protein.
Results: Considering the general formula (I) of the iminodiacetic (IDA) mono-hydroxamate derivatives, several substituent groups were tested. The introduction of the 4-sulfamoylphenylethyl (SPE) moiety as R2 group lead to a general increase of activity with most MMPs but, more significantly, endowed with the ability of inhibiting CAs. The apolar isopropyl group in -position to the hydroxamic group also reflected an increase of inhibitory activity with both MMPs and CAs.
 
Comp.
|
R
|
R2
|
IC50 (nM)
|
KI (nM)
|
MMP-1
|
MMP-2
|
CA II
|
CA IX
|
2
|
H
|
OH
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1.53 ×103
|
1.2
|
-
|
-
|
16
|
H
|
SPE
|
143
|
1.57
|
65
|
12
|
20
|
i-Pr
|
SPE
|
7.8
|
0.35
|
4.2
|
3.3
|
Docking studies revealed interaction of the SPE group with conserved residues of S2’-S3’ subsites of the MMPs, this being the reason for the high affinity and low selectivity of these compounds with this family of metalloenzymes.
Conclusions: This work demonstrated that the introduction of the
4-sulfamoylphenylethyl group in the IDA scaffold maintains the activity on MMPs, providing a good activity also on some CAs. This finding opens the way to further studies directed to the identification of dual inhibitors selective for those enzymes implicated in cancer, such as MMP-2 and CA IX.
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Can We Make Eucaryotic Cells Resistant To Antibiotics? Correlation Between Multidrug Resistance-Associated Protein (Mrp) Efflux Pump Expression And Fluoroquinolones Accumulation In J774 Macrophages
MARQUEZ B, VALLET C, MINGEOT-LECLERCQ MP, TULKENS PM, VAN BAMBEKE F
Unité de Pharmacologie cellulaire et moléculaire, Université catholique de Louvain, Brussels, Belgium
Background: Over-expression of multidrug transporters is a well-known mechanism of resistance to anticancer agents in eucaryotic cells, and to antibiotics in prokaryotic cells. In vitro, their overexpression can be obtained by prolonged exposure to drug substrates. We have examined here whether chronical exposure to fluoroquinolone antibiotics could trigger a phenotype of resistance in eucaryotic cells as well, based on our previous observation that ciprofloxacin (CIP) is subject for an active efflux by an Mrp–like (Multidrug resistance-related Proteins) transporter in J774 macrophages (M), which decreases its accumulation and activity against intracellular bacteria, while moxifloxacin (MXF) was not affected (AAC 2005, 49:2429-2437).
Methods: J774 M were exposed for several months to increasing concentrations in either ciprofloxacin or moxifloxacin (from 17 to 68 mg/L). CIP and MXF accumulation was measured by fluorimetry in the resulting cell lines as compared to wild-type M. Mrp expression was evaluated by real-time PCR and Western-blot.
Results: As compared to wild-type M, which accumulate CIP about 4-fold and extrude it with a t1/2 ~ 1.2 min, cells exposed to CIP showed a markedly decreased accumulation of CIP (< 1-fold) related to a faster efflux (t1/2 < 0.1 min), while cells exposed to MXF showed a higher accumulation of CIP (15-fold) and a slower efflux (t1/2 ~ 2.6 min). MXF accumulation was high (15-fold) and similar in all cell types.
Analysis at the mRNA and protein levels revealed an overexpression of Mrp2 and Mrp4 in M exposed to CIP and a reduction of Mrp4 expression in M exposed to MXF.
Conclusions: Exposure of J774 M to the Mrp substrate CIP selects for a resistant phenotype characterized by the overexpression of two Mrp transporters. Exposure of the same cells to the non-substrate MXF selects for an 'anti'-resistant phenotype, with reduction in the expression of Mrp4. These data suggest that fluoroquinolones can differentially affect the expression of Mrp transporters in J774 M, illustrating the complexity of the interactions between closely-related drugs and transporters.
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Success Story Of The First Regulatory Approval Of Safety Biomarkers, Part I: From Identification To Biological Qualification
MARRER E, DIETERLE F
Novartis Institutes for BioMedical Research, Basel, Switzerland
Background: In the context of the Critical Path Initiative, a project on the validation and qualification of new renal safety biomarkers has found a successful closure.
Methods: The project focused on rat nephrotoxicity, whereby 8 known nephrotoxicants and 2 known hepatotoxicants were administered at different dose levels to assess diverse modes and severity grades of toxicity. To enrich the set of functional markers (serum creatinine and blood urea nitrogen) used for the last hundred years, gene expression profiling was performed in the kidney and correlated to the histopathology.
Results: Transcriptomics allowed the identification of 15 promising new injury biomarkers covering toxicity in proximal tubules, distal tubules, and glomerules. These 15 biomarkers were then used to monitor changes of their protein levels in kidney, blood and urine.
This project also allowed the establishment and testing of the implementation of an early safety biomarker validation process. The first step of the process was the discovery phase and selection of exploratory biomarkers, model compounds and the definition of the animal model. Then, appropriate analytical methods (multiplexed immunoassays and RT-PCR) were developed and validated. The in-life phase started with a dose-range finding study followed by the main validation studies comprising of 960 animals in total. In these multiple models of kidney injury, the sensitivity and specificity of the new markers were assessed and compared to the “gold standards” serum creatinine and blood urea nitrogen.
In addition, a gene expression atlas across multiple organs in baseline and treated animals was compiled for the best performing markers.
Conclusions: A subset of these markers demonstrated higher diagnostic performance than serum creatinine and blood urea nitrogen, especially for low-grade tubular and glomerular injury. These data were submitted to the FDA and EMEA as first Voluntary eXploratory Data Submission (VXDS) and subsequently 5 urinary biomarkers were approved for use as biomarker to monitor renal injury in pre-clinical studies.
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