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Forschung und Berichte in den medizinischen Wissenschaften

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Volumen 4, Ausgabe 3 (2020)

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Global Dermatology 2018 -Topical Nutlin-3 Potentiates the UVB-induced p53 Response and Reduces DNA Photodamage and Apoptosis in Mouse Epidermal Keratinocytes in Vivo- Birthe Mørch Thomsen- Copenhagen University

Birthe Mørch Thomsen

Background/Aims: (-)-Nutlin-3 (nutlin) may be a cis-imidazoline analog, which activates p53 by antagonizing murine double minute (Mdm2) protein. We therefore conducted a study in hairless mice to analyze the effect of topical nutlin on murine epidermis after UVB-radiation.

Methods: Animals were euthanized 24 hours after irradiation, the dorsal, treated skin was biopsied and fixed in 4% formalin. Sections were incubated with the antibodies against p53, thymine dimers and terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling (TUNEL).

Results: We showed here that nutlin was active after topical application in hairless mice and potentiated the p53 nuclear translocation in epidermal keratinocytes after ultraviolet B irradiation. Moreover, topical treatment with nutlin resulted in a decrease in the number of keratinocytes exhibiting positive nuclear staining for thymine dimers (P<0.05compared with the vehicle control) and decreased the frequency of apoptotic, TUNEL-positive cells (P=0.02).

Conclusion: We hypothesize that nutlin stimulates DNA photodamage repair in the epidermis and may prove useful for the chemoprevention of skin cancer.

Keywords: Nutlin-3; p53; Apoptosis; Thymine dimer; Hairless mice

Experimental Design:

 Animals: Female C3.Cg/TifBomTac immunocompetent mice (age 15 weeks) were purchased from Taconic (Ry, Denmark). The mice were kept in an animal facility on a 12 h light/dark cycle at 23 - 24°C. Animal care and treatment followed Danish national guidelines.

Drug treatment, ultraviolet source and experimental design :

Mice were sedated with 0.05 ml HypDorm (fentanyl citrate 0.158 mg/ml, fluanisone 5mg/ml, midasolam 2.5mg/ml) and treated on one of the lateral halves of the back skin with 100 µl isopropanol solution of 43 mM nutlin [(-) 4-(4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4- methoxyphenyl)-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-2- one] (Cayman Chemical) 30 min before irradiation. The other half was treated with the same volume of isopropanol. The mice were then irradiated with 3 SED (100 mJ/cm2 ) UVB from a light source comprising an array of 6 TL12 tubes (Philips, Eindhoven, The Netherlands) and re-treated with nutlin or isopropanol, as above. Control mice were not irradiated. Animals were euthanized 24 hours after irradiation because studies in vitro in human skin and in vivo in mice skin suggest that after 24 hours about half of the CPD are repaired [5,6]. The treated dorsal skin was biopsied and fixed in 4% formalin.

Immunohistochemistry :

4 µm sections were cut from the paraffin embedded skin biopsies, deparaffinised with xylene, re-hydrated and incubated with the antibodies against p53 (rabbit, polyclonal, Novocastra Newcastle upon Tyne, UK) or peroxidase-conjugated monoclonal anti-thymine dimer antibody (Kamiya Biomedical, Seattle, WA). The antibodies were visualised using the LSAB+ System-HRP (Dako, Glostrup, Denmark). Terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling (TUNEL) was performed using the DeadEnd Colorimetric TUNEL system (Promega, Madison, WI). Apoptotic, TUNEL-positive cells were counted in a 3 mm long section of epidermis. The extent of thymine-dimer positivity was assessed on an ordered categorical interval scale: 1) 0% stained nuclei, 2) 1-25%, 3) 26-50%, 4) 51-75% positive nuclei. In no samples the frequency of positive nuclei exceeded 75%.

Results :

As shown in Figure 1, the p53 staining of unexposed epidermis revealed moderate immunoreactivity in the cytoplasm but not in the nuclei of epidermal keratinocytes. After UVB irradiation the cytoplasmic staining was more intense and single cell nuclei were stained positive for p53. In non-irradiated skin nutlin did not induce p53. Since it is known that p53 is induced by DNA photoproducts and p53 is involved in the repair of CPD [7] we stained skin biopsies with the antibody against thymine dimers. Topical treatment with nutlin resulted in an overall decrease in the number of keratinocytes exhibiting positive nuclear staining for thymine dimers.Nutlin treatment significantly inhibited the UVB-induced apoptosis (Figure 2C-D). TUNEL staining revealed 9.0 ±2.6 (mean ±standard deviation) TUNEL-positive cells / mm epidermal length in UVB-irradiated, vehicle-treated skin versus 1.7 ±0.5 TUNEL-positive cells /mm in the nutlin-treated skin (P=0.02, paired t-test).

Conclusions :

This study shows that the Mdm2 inhibitor, nutlin, activates p53 in the epidermis in UVB-irradiated mice. This was accompanied by a significant decrease in the frequency of the cells harbouring thymine dimers and diminished keratinocyte apoptosis. We suggest that the decreased apoptosis is caused by enhanced CPD repair due to p53 activation by topically applied nutlin. It is conceivable that nutlin may be used for chemoprevention of squamous cell carcinoma in humans.

Conflicts of Interest :

This study was financed solely by the Bispebjerg University Hospital and has not been supported by any pharmaceutical company.

Bottom line: This work is partly presented at 17th European Dermatology Congress March 01-03, 2018 | Paris, France.

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Hematologists 2019- Chronic Neutrophilic Leukemia: A Rare and Difficult Diagnosis of Exclusion- James Ziai- Yale University School of Medicine

James Ziai

Chronic neutrophilic leukemia (CNL) is very  rare myeloproliferative disorder that presents diagnostic challenges for both pathologists and treating clinicians. Because this disease entity is extremely rare, and because it’s typically a diagnosis of exclusion, its important for pathologists and hematologists to be accustomed to CNL when approaching the patient with a myeloproliferative clinical picture. Thus, the objectives of this report are: 1) to detail the clinical case of a 59 year old male veteran with initial presentation of hyperleukocytosis, 2) to review the differential diagnosis of a granulocytic myeloproliferative presentation and demonstrate the laboratory and clinical criteria utilized to establish a diagnosis of CNL during this case, and 3) to briefly review the current literature on the diagnosis and treatment of CNL

Keywords: Chronic neutrophilic leukemia; A typical chronic myelogenous leukemia; Myeloproliferative diseases

Introduction:

 Chronic neutrophilic leukemia (CNL) could be  a rare myeloproliferative neoplasm characterized primarily by leukocytosis, but often lacking distinct clinical, laboratory, and molecular features. Assessing the patient with an atypical myeloproliferative picture and correctly making the diagnosis of CNL can be challenging for pathologists and clinicians alike. The aims of this report are to detail the clinical case of a 59-year-old male veteran with initial presentation of hyperleukocytosis so as to demonstrate the laboratory and clinical criteria utilized to establish a diagnosis of CNL. We also briefly review the current literature on the diagnosis and treatment of CNL.

Case Report:

A 59-year-old male veteran presented to our facility (VA Connecticut Healthcare System, West Haven, CT, USA) with chief complaints of shortness of breath (SOB) and fatigue of approximately two weeks duration. The patient had been diagnosed with hypertension in the past and was taking amlodipine, labetalol, and aspirin. He had no relevant case history and was a non-smoker. On review of systems the patient denied wheezing, cough, orthopnea, syncope, chest pain, fever or any particular exacerbating or remitting factors for his current condition. Pertinent physical exam findings included a lethargic appearance, mild splenomegaly, and a 2x2x1cm fixed painless, hard mass on the right anterior mandible.

Imaging studies were also performed to investigate for the possibility of other malignant or reactive causes for the patient’s leukocytosis. Chest x-rays, performed on admission and on several days during the course of the patient’s hospital stay, showed some evidence of pulmonary congestion consistent with mild congestive heart failure, but without evidence of an infectious infiltrate or malignant process. A CT scan of the thorax performed on day 4 of admission showed mild, “non-specific” mediastinal adenopathy, without other significant findings. Retroperitoneal ultrasound demonstrated echogenic kidneys and a 4 cm hepatic cyst cluster, but no other significant abnormal findings. In the absence of any evidence for a reactive cause of the leukocytosis, a bone marrow aspirate and biopsy were performed. The bone marrow core biopsy was markedly hypercellular for age with a cellularity estimated at 90% (Figure 1a). Myeloid maturation was sequential with a predominance of mature granulocytes, mainly localized in interstitial areas (Figure 1b and 1c). proliferation but otherwise appeared normal and megakaryocytes were mildly increased with normal morphology .There was no relative increase in myeloblasts, eosinophils, basophils, or mast cells. Moderate focal reticulin fibrosis was noted in paratrabecular regions, but didnt reach  to cortical areas. The bone marrow aspirate reiterated the core biopsy findings. There was toxic granulation noted on neutrophils and a few          hypersegmentation noted on peripheral smear.

Discussion:

 Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by proliferation of one or more elements of the neoplasms associated with abnormalities of PDGFRα and PDGFRβ were also essentially ruled out by negative molecular testing. Second, neutrophil precursors make up less than 10% of circulating leukocytes. Finally, the patient’s chronic, relatively indolent clinical course is consistent with most reports of CNL as opposed to atypical CML which often follows a rapid and aggressive downward course. In summary, this is a case of a 59-year-old man presenting with SOB and hyperleukocytosis ultimately diagnosed as CNL. This case demonstrates the issue in firmly establishing a diagnosis of CNL and the way clinical and laboratory criteria may be utilized to discriminate between similar, but distinct, disease entities. In this case, a diagnosis of CNL not only influenced the initial treatment choice, but also provided important prognostic implications. A lack of reproducible molecular abnormalities remains a major hurdle in clearly identifying CNL cases. Future work in characterizing the molecular profiles of this and other rare myeloproliferative neoplasms would aid not only in their classification and diagnosis, but could also yield novel treatment options.

Acknowledgments :

 The authors wish to acknowledge the assistance of Dr. Nelofar Shafi for this case. This material is the result of work supported with resources and the use of facilities at the VA Connecticut Healthcare System.

Bottom line: This work is partly presented at 16th World Hematology Congress March 18-19, 2019 Rome, Italy

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Euro Biosensors 2018- Using Nano-Arrayed Structures in Sol-Gel Derived Mn2+ Doped Tio2 for High Sensitivity Urea Biosensor- Ravi R. Pandey- National Physical Laboratory

Ravi R. Pandey

Ordered and self-organized nano-array structures have been developed by Mn2+ doping in TiO2 thin fi lms deposition on conducting substrates by dip coating technique. Mn doped TiO2 thin fi lms exhibits better bioactivity for enzyme immobilization and cyclic voltammetry measurement has been used for qualitative characterization of electrochemical induction of oxidation-reduction process in TiO2 and Mn doped TiO2 fi lms. Due to presence of Mn2+ ions at fi lm surface, current voltage characteristic of Mn doped TiO2 matrix was enhanced by a factor of ten and it had also reduced the crystallite size and promoted transformation of anatase to rutile phase of TiO2 . Urea concentration in the electrolyte was determined by observing chronoamperometry response on urease immobilized working electrodes. The urea detection sensitivity of the Mn doped TiO2 thin fi lms base platform was 2.3 μA mM−1 cm−2 which is about 15 times higher from only TiO2 base platforms. Such kind of enzyme–TiO2 /Mn nano-array electrode could contribute a potential prospect in low cost biomedical diagnosis..

Keywords: Nano-array structures; Mn doped TiO2 ; Chronoamperometry; Cyclic voltammetry

Introduction:

Application of nanoscale materials for electrochemical biosensors has been grown exponentially due to high sensitivity and fast response time . These factors eventually controlled by the interaction effectiveness and determine selectivity and sensitivity of materials for potential application in the area of biomedical diagnosis . Extensively TiO2 and TiO2 with different polymer matrixes have been used for different applications such as DNA biosensor, enzyme immobilized platforms, detection of Pseudomonas aeruginosa and detection of toxic compounds . Stability and usefulness of structural modification for efficient immobilization of biomolecules in TiO2 nanotube arrays based biosensor has been also discussed . It has been demonstrated that the large surface area, good chemical stability and nontoxicity of the TiO2 have been achieved with different nanocomposite and 3D macroporous structures . Electrochemical synthesis of titanium oxide nanotubes (TiO2 ) in different types of electrolytes and applications for biosensors applications has showed great interest . The length and pore diameter of the TiO2 layers have been tailored by varying electrolyte composition, applied potential, pH, and anodizing time . We report a new strategy for Mn2+ induced nano-arrayed structures in sol-gel derived TiO2 platforms for biosensing applications. Previously, anatase–rutile phase transformation of TiO2 in sol–gel method synthesized has been achieved by various amount of manganese (Mn) ions and has been reported to be among the most efficient transition-metal oxide catalysts for catalytic disposal of pollutants . However, using Mn doping with TiO2 for biosensor applications has not been explored.

Experimental:

0.25 mole of HNO3 was added to alcoholic solution of 0.5 M Ti(OBu)4 in 1:2 molar ratio of HNO3 : Ti(OBu)4 . After that 0.5 mole water was added to this solution drop wise using dropping funnel in 1:1 molar ratio of H2 O: Ti(OBu)4 . A clear yellowish, transparent and stable TiO2 colloidal sol was obtained. A stock solution of 0.5M Manganese (II) acetylacetonate (Mn(acac)2) was prepared in isopropanol. To prepare 5 mole % Mn2+ ion doped TiO2 sols of stock solution was added respectively to TiO2 sol. After 24 hours, the above solution used to coat the thin films by dip-coating technique at constant pulling speed 25cm/min on 2x4cm2 ultrasonically cleaned glass substrates under controlled relative humidity (30-40%) condition and room temperature. These samples were further dried at 100°C in electric oven for 60 minutes followed by annealing at 550°C for five hours in programmable furnace. All chemicals were analytical grade and purchased from Sigma Aldrich otherwise were specified. The TiO2 and TiO2 /Mn electrodes were immersed in 50 g L−1 urease prepared in 0.1M phosphate buffer solution for 12 h at 4°C. The enzyme immobilized electrodes were washed with phosphate buffer solution to remove unbounded sites.

Result and Discussion:

0. 2θ values of diffraction peaks observed in XRD spectra of undoped TiO2 were at 25.28, 37.78, 48.02, 53.92, 55.06, 62.72, 68.80, 70.36, and 75.12. These peaks were assigned to reflections from (101), (004), (200), (105), (211), (204), (116), (220), and (215) crystal planes, which mainly corresponds to anatase phase of TiO2 . Prominent diffraction peaks of 5 mole % Mn2+ doped TiO2 films 2θ values were at 25.41, 27.60, 36.13, 37.80, 39.24, 41.29, 44.13, 48.13, 54.46, 56.79, 62.59, 64.14, 68.79, and 75.12. These peaks were assigned to reflections from (101A), (110R), (101R) (004A), (200R), (111R), (210R), (200A), (211R), (220R), (204A), (310R), (116A), (215A) crystal planes, which corresponds to both anatase and rutile phase of TiO2 and notation ‘A’ and ‘R’ were associated with anatase and rutile. After Mn2+ doping in TiO2 the mix phase of anatase and rutile was obtained and a small shift in 2θ value was also observed. For analysis of particle size from XRD peak broadening, the strongest peak i.e. 101 was chosen and Debye-Scherrer’s equation was used for calculation. The calculated value of average crystallite size in pure & 5 mole % Mn doped TiO2 samples is 28±2 nm & 20±2 nm respectively. XRD studies showed decrease in crystallite size induced by the dopant ion, therefore more surface free energy in case of doped samples. Secondly, the incorporation of foreign atoms in a lattice of TiO2 can create defects during formation of original crystal lattice. These results agree with other reported work in literature on effects of manganese to accelerate anatase-to-rutile transformation [19]. The most common effect of incorporation of foreign atoms in a lattice is the defect formation in the original crystal lattice which could develops regular patterns of nano-structures.

Conclusion:

Mn induced nano-arrayed structured TiO2 platform has been developed by simple sol-gel process and dip coating techniques. Such types of platforms has demonstrated usefulness for biosensor which could provide more effective interaction areas and more feasible electron transfer interfaces to support amperometric response of electrode. On the base of enzyme catalysis and electrochemical reduction reaction under a potentiostatic condition, the biosensing application was achieved by applying the uresae–TiO2 /Mn electrode. This biosensor exhibited a very high sensitivity and low detection limit for urea detection. Such kind of enzyme–TiO2 /Mn nanotube array electrode could contribute a potential prospect in low cost biomedical diagnosis.

Bottom line: This work is partly presented at 9th Euro Biosensors & Bioelectronics Congress November 29-30, 2018 Dublin, Ireland

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Addiction Summit 2018- Methamphetamine Cured my Cocaine Addiction - Colin N. Hail- Baylor College of Medicine

Colin N. Hail

Three methamphetamine-dependent participants that had been in our clinical laboratory experiments and previously obsessed on cocaine are reviewed.

Keywords: Drug abuse, Stimulants, Tolerance, Pharmacotherapies, Prodrug

Introduction:

 Cocaine increases central catecholamines that are linked to its positive subjective and reinforcing effects . Drugs with similar pharmacological action to it of cocaine like d-amphetamine (AMPH) and medically formulated methamphetamine (METH) administered chronically produce tolerance to its reinforcing effects and   should be useful in treating cocaine dependence.Accordingly, recent clinical studies show that AMPH attenuates cocaine’s positive subjective effects  and that treatment with sustained-release METH reduces rates of cocaine positive urine and decreases craving for cocaine in cocaine-dependent individuals. Consistent with these studies, we review three unique case histories of METH-dependent participants previously obsessed on cocaine who abruptly stopped once they started using METH. In essence, they stated that METH “cured” them of their drug addiction.

Materials and Methods:

A detailed demographic and drug use history was obtained during a basic screening interview for admission into one of our clinical laboratory trials. Participants tested positive for METH on initial screening yet none demonstrated overt clinical signs of METH intoxication. Participant primary and secondary screens were reviewed. Inquiry was aimed at why they thought METH may have helped them and what new adverse consequences they may have experienced by abusing another highly addictive substance.

Results:

Participant 1 (P1):

P1 was a 40 year-old male Caucasian of Philippine descent who used cocaine and alcohol heavily before 2005. By his own estimation he was “severely” obsessed to cocaine and alcohol. Addiction to these substances directly contributed to numerous adverse interactions with law enforcement and irreparable harm in relations with his family and friends.. He described cocaine because the “ultimate evil” that brought out the “hater” in him. Depression, suicidal ideation, financial stress and threats of violence from cocaine dealers forced him to leave his home. Soon after he moved to Houston, he was given METH at a party and felt it had “benefitted his being”.. He stated emphatically that he had not used, nor had any searching for cocaine or alcohol, since first starting METH even when given the chance to use these substances. During screening, he reported that he used METH 25 days out of the 30 before  to the interview and no cocaine or alcohol for 4 years. Similar to consequences he suffered when abusing cocaine, he continued to own financial and legal problems associated with  his heavy METH intake. In contrast to his past cocaine and drug  abuse, METH had no effects on his daily nicotine (1 pack per day) and cannabis (2 joints per day) use. In fact, P1 indicated that cannabis, but not nicotine, increased both the ‘high’ and ‘desire’ associated with using METH.

Participant 2 (P2):

P2 was a 40 year old, Caucasian male musician who had some success touring and recording with various popular bands in the 1980s and 1990s.  According to P2, METH allowed him to be more productive even though he felt he rarely finished any projects he initiated. His last gainful employment was in 2009 teaching guitar lessons which he left likely due to paranoia secondary to METH. Similarly, P2 stated that the reason he left his last band was because the leader: “kept giving me that evil eye, ya know, when they look at you like that, it’s a bad sign.  P2’s responses on multiple drug, use questionnaire indicated that cannabis reduced the ‘high’ associated with METH and decreased ‘desire’ for the drug whereas nicotine had not effect.

Participant 3 (P3):

P3 was a 30 year old Hispanic male, who had been using METH for 7 years. Similar to the participants reviewed above, P3 enjoyed METH’s cognitive effects and felt he was more productive yet he always needed money to support his large METH intake (approximately 1 gram per day). He was inconsistently employed and held numerous jobs often circumventing resources from them clandestinely. He also stated that METH enhanced his sexual performance, which was important to him since he worked in pornography.

Discussion:

These cases appear to support preclinical and clinical studies suggesting that chronic treatment with AMPH-like stimulants—such as METH—may be efficacious for cocaine dependence. Interestingly, each participant’s searching forcocaine was abolished once they began using METH. This could suggest that they are likely replacing one addictive substance for another similar to methadone maintenance therapy for opioid dependence. Still, once they started using METH, their searching for cocaine was abolished. This anecdote is according to recent controlled clinical studies showing that chronic treatment with AMPH blocks cocaine’s positive subjective effects and sustained-release formulations of METH decrease cocaine use in humans ..AMPH and METH are not viable treatments for cocaine dependence due to high abuse liability and obvious adverse social, financial and psychiatric consequences associated with use of these stimulants as demonstrated by the cases described above. Medications used to treat attention deficit hyperactivity disorder in children and adolescents in sustained – release formulations may be a better option. Also, stimulant pro-drugs like lisdexamfetaminedimesylate that’s activated only after its metabolized may offer a viable option since their inherent slow onset of action and entry into the CNS reduces abuse liability . Our research group is currently planning laboratory studies assessing stimulant pro-drugs as potential medications for cocaine dependence.

Bottom line: This work is partly presented at 8th International Conference on Addictive Disorders and Alcoholism May 17-18, 2018 Singapore

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Human Genetics 2019- Progesterone inhibition of MDM2 p90 protein in MCF-7 human breast cancer cell line is dependent on p53 levels - Moussa Alkhalaf - Kuwait University

Moussa Alkhalaf

We present evidence that reestablishing of P53 expression by transient transfection of P53 cDNA in these cells enhances the expression level of MDM2 p90 isoform. Chronic neutrophilic leukemia (CNL) could be  a rare myeloproliferative neoplasm characterized primarily by leukocytosis, but often lacking distinct clinical, laboratory, and molecular features. Assessing the patient with an atypical myeloproliferative picture and correctly making the diagnosis of CNL can be challenging for pathologists and clinicians alike. The aims of this report are to detail the clinical case of a 59-year-old male veteran with initial presentation of hyperleukocytosis so as to demonstrate the laboratory and clinical criteria utilized to establish a diagnosis of CNL. We also briefly review the current literature on the diagnosis and treatment of CNL.

Keywords: MDM2, isoforms, p53, p90, progesterone, MCF-7, breast cancer

INTRODUCTION:

The mdm2 gene was originally cloned as an amplified gene on a murine double-minute chromosome in the tumorigenic 3T3DM murine cell line (Fakharzadeh et al, 1991). MDM2 expression is controlled at the transcriptional level from P53-independent (P1) and P53-responsive (P2) promoters (Zauberman et al, 1995), both encoding a 90 kDa full length MDM2 (p90) protein (Brown et al, 1999). In addition, MDM2 proteins of smaller sizes have been identified (Olson et al, 1993Perry et al, 2000Bartel et al, 2002). These differently sized proteins arise through either proteolytic cleavage (Pochampally et al, 1998), internal translational initiation (Saucedo et al, 1999) or alternative splicing (Sigalas et al, 1996Matsumoto, 1998). Although the biochemical functions of these small proteins have not yet been determined, the MDM2-p90 isoform binds to and inactivates P53 tumor suppressor protein suggesting that MDM2 can function as a negative feedback regulator of P53 (Momand et al, 1992Barak et al, 1993). Lukas et al (2001) suggested that MDM2 expression is altered in invasive breast cancer and is associated with more aggressive disease. We have recently demonstrated that the progesterone-induced growth inhibition of the MCF-7 human breast cancer cell line was associated with down-regulation of P53 endogenous levels (Alkhalaf and El-Mowafy, 2003). Because the regulation of MDM2 expression by P53 has been proposed by several authors to be the mechanism by which P53 balances its own activity (Juven et al, 1993Midgley and Lane, 1997Prives, 1998), we hypothesized that the decrease in P53 levels seen in MCF-7 cells treated with progesterone would affect MDM2 expression. We report here that in MCF-7 human breast cancer cells treated with progesterone, MDM2 p90 but not MDM2 p57 is down-regulated.Overexpression of P53 in MCF-7 cells stimulated the MDM2 expression and abrogated the effect of progesterone.

MATERIALS AND METHODS:

Cell lines and culture conditions:

The breast cancer cell lines MCF7, T47D, and MDA-MB231 were kindly provided by Bohdan Wasylyk (IGBMC Core Facility, Strasbourg, France). The MCF-7 cells contain functional P53 protein localized at the nucleus (Wasylyk et al, 1999) and classified as progesterone and estrogen receptor positive. The T47D cells have a mutated type of P53 which is localized in the cytoplasm (Schafer et al, 2000) and contain both estrogen and progesterone receptors. The cells were grown in RPMI1640 medium (Gibco BRL) supplemented with 5% fetal bovine serum, glutamine and gentamicin and maintained in a 5% CO2 humidified atmosphere in a 37 °C incubator.

Western Blot Analysis:

Cells were washed twice with PBS buffer then the preheated (95°C) lysis buffer [20 mM Tris-HCl pH 7.4, 20 mM dithiothreitol (DTT), 2 mM EDTA (sodium salt), 1% (v/v) Triton X-100, 1% (v/v) NP40, 1% (w/v) sodium deoxycholate, 1 mM sodium pyrophosphate, 1 mM sodium orthovandate (prepared in Tris buffer) and 1 mM phenylmethylsulphonyl-fluoride] was added directly to the cell monolayer. The cells were scraped and mixed with a rubber policeman, transferred to Eppendorf tubes and centrifuged at 13000 x g for 5 min. The resulting supernatant was saved and the protein was determined by the Bradford method. Extracts were boiled for 3 min in 2 x SDS buffer. Equal amounts of protein were loaded on 10% (w/v) polyacrylamide gels according to the method of Laemmli and then electrotransferred onto nitro-cellulose membranes. The blots were incubated first with anti-MDM2 (Ab-1, clone IF2) monoclonal antibody (Oncogene Research Product, Calbiochem). The antibody is directed against the N-terminal fragment between amino acid residues 26-169 of human MDM2 (Fakharzadeh et al, 1991) then incubated with peroxidase-conjugated anti-mouse IgG (Jackson Laboratory, USA) at 1/2000 dilution. Immunoreactive bands were visualized by incubation with luminol (according to manufacturer's instructions; ECL Western blotting detection system from Amersham). The blots were stripped and hybridized with P53 (DO1) monoclonal antibodies (Oncogene Research, Cambridge, MA, USA) and processed as for the MDM2 antibodies. The TATA- Binding Protein (TBP) monoclonal antibody was used as a loading control (kindly provided by IGBMC Core Facility, Illkirch, France). Another MDM2 antibody was used, MDM2 (C-18) purchased from Santa Cruz Biotechnology (California, USA). It is an affinity purified rabbit polyclonal antibody raised against a peptide mapping within the carboxyl terminal domain of MDM2 of human origin. A prestained SDS-PAGE molecular weight protein standards (low range) is used to estimate the molecular weights of proteins ((Bio-Rad, UK).

RESULTS:

The expression of MDM2 proteins in MCF7, T47D, and MDA-MB231 human breast cancer cell lines was analyzed by Western blotting with anti-MDM2 (Ab-1) monoclonal antibody.  shows that, with this antibody, four MDM2 isoforms were detected, the p57, p76, p80, and p90 kDa in all three cell lines. A prominent expression of MDM2 p57 protein as compared to the p90, p80 or p76 was observed in the three cell lines used in this study. In MCF-7 cells that contained functional P53 protein, the endogenous MDM2 p90 protein level was higher than these in T47D and MDA-MB 230 cell lines (both have mutated P53 proteins). In contrast, MDM2 p57 appeared to be lower in MCF-7 as compared to its levels in the cells which have mutated P53. The high level of MDM2 p57 was observed in other breast cancer cells, a normal breast cell line and in other types of cancer cell lines .

Bottom line: This work is partly presented at 8th International Conference on Human Genetics and Genetic Diseases November 25-26, 2019 Madrid, Spain, USA

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HealthCare 2017- Laparoscopic Gastric Bypass in a Patient with Peritoneal Encapsulationand Malrotation of the Intestine - Carmen Mesas Burgos- Bariatric Center Stockholm

Carmen Mesas Burgos

 The number of bariatric procedures performed each year has increased exponentially worldwide. Obesity is increasing at an alarming rate in the developed world, reaching epidemic proportions. In Europe one fifth of the population is obese [1]. Several studies have shown a significant weight loss in patients treated surgically, with a long-term maintenance of the weight loss and improvement in many of the obesity-related medical illnesses [2]. Peritoneal encapsulation is a very rare developmental malformation, characterized by the presence of an accessory peritoneal sheath covering part or the entire small bowel. Clinically, most of the cases are diagnosed as incidental findings during unrelated surgery, and only few presented as bowel obstruction [3-6]. The incidence of colon malrotation in adults has been estimated to be 0.2-0.5 % in older reports .the majority are asymptomatic; found incidentally at the time of another operation or radiologic examination, and very few will become clinically evident .

Case Report:

A 38 years old man with a long-standing history of morbid obesity and body mass index of 35.5 kg/m2 was schedule for laparoscopic Roux-en-Y gastric bypass and met the International Federation for the Surgery of Obesity, IFSO, consensus criteria for bariatric surgery [11]. Preoperative assessment was performed and the patient underwent surgeon, dietitian, nurse and anesthesiologist consults, and routine blood tests were run. The patient did not have other associated comorbidities or previous medical history, and no other preoperative radiological examinations were performed. He denied any abdominal complaints of pain, nausea, vomiting or constipation, and had no past surgical history. The patient underwent schedule LRYGB. The surgery was started routinely in French position, with the surgeon standing between the patient’s legs with the patient in reverse Trendelenburg position. The procedure was performed using four 12 mm trocars and a Nathan’s retractor to lift up the liver. the remainder trocars were placed over the right upper quadrant, midline, subxiphoid and left flank regions under direct laparoscopic.To identify the Treitz ligament, the proximal small intestine was not found in its usual location at the base of the transverse mesocolon. Instead, a peritoneal membrane was found at the place of the duodenaljejunal flexure. Despite extensive exploration, the anatomy could not be clearly defined, and an extra 12-mm trocar was placed in the left lower quadrant. The accessory peritoneal membrane lied posterior to the greater omentum, separating it from the small intestine. The colon and ileo-cecal valve were found at the normal position.

Discussion:

With the increasingly number of gastric bypass procedures for treatment of morbid obesity, unexpected challenges might encounter. This case highlights the need for bariatric surgeons to have full understanding of congenital anomalies and their management. Understanding the embryology and development of the midgut is essential in understanding and treating rotational defects of the intestines. The primitive digestive tube in the early embryo is a straight tube that consists of the foregut supplied by the celiac artery, the midgut supplied by the superior mesenteric artery (SMA), and the hindgut, supplied by the inferior mesenteric artery. a process divided in three stages: the first stage starts in the fifth g.w., when the midgut’s cranial limb will rise to the distal portion of the duodenum, jejunum and major portion of the ileum. the orientation of the limbs in the creation of the entero-enterostomy: laying the bilio-pancreatic limb towards the patient’s right side instead of the left, and the alimentary limb towards the left side instead of the right side like in the standard procedure, is the best orientation to prevent internal hernia, kinking and rotation. Moreover, a clear understanding and visualization of the anatomy are mandatory, and extra trocars should be widely used. In our case, the colon and caecum where placed in their normal position in the abdomen, no Ladd´s bands were found, but the Treiz’s ligament was rudimentary and the duodenal-jejunal junction was placed in the right upper quadrant. Both malformations take place in the same time frame of embryologic development, at 12th g.w.

Bottom line: This work is partly presented at Joint Conference on 6th International Conference on Clinical & Medical Case Reports & 11th Global Healthcare & Fitness Summit October 16-18, 2017 San Francisco, USA

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Breast Cancer 2018-A Comparison Between Oncoplastic Breast Conserving Surgery andStandard Wide Local Excision: A UK Experience- Saira Khawaja- Prince Philip Hospital

Saira Khawaja

 A total of 319 consecutive patients were included in the study. The majority of those latter surgeries involved volume displacement methods. Demographic data was collected. Statistical analysis was performed using SPSS software. Our results demonstrated that OPS excised significantly larger tumors (p (p<0.05) of greater weights(p<0.05) with little cosmetic penalty. There was no significant difference in margin clearance (p = 0.10) or the number of patients requiring further surgery in both groups (p=0.254).OPS provides a better cosmetic and oncological result compared to standard wide local excisionsbased on the weight and size of the tumor. Margin clearance is comparable in both groups.

Keywords:

Breast conserving surgery; Oncoplastic breast surgery; Wide local excision; Breast cancer; Cosmesis

 

Introduction:

In women with early stage unifocal carcinoma, breast conserving surgery followed by radiotherapy is the recommended choice. However, in some women, lesions are difficult to excise without the danger of cosmetic deformity or inadequate margin clearance. These women often present with large tumors in relatively small breasts requiring an excision of roughly 15% to twenty% of the breast volume or over  30% in large breasts. Another factor affecting poor cosmesis after breast conserving surgery is tumors located in aesthetically sensitive areas such as the central, medial, and inferior quadrants.. OPS combines the principles of oncologic and cosmetic  surgery techniques to gain oncologically and aesthetically pleasing results . As these techniques become more accepted there is a demand for surgeons to become familiar with the indications and skills required to make oncoplastic surgery safe and effective . The choice between different oncoplastic techniques are determined mainly by the location  of the tumor in the breast, tumor characteristics, extent of resection, breast characteristics (size, shape and glandular density), previous surgery, and the expectations and wishes of the patient.

 

Patients and Method:

Patients were not matched for age. Those who underwent a standard WLE included a total of 265 consecutive patients whilst 54 in number underwent OPS. The symptomatic and screening populations with a diagnosis of invasive cancer or DCIS were included. All patients were operated on by a team of breast surgeons. Appropriate axillary surgery was carried out at the time of the initial procedure. Intraoperative sentinel lymph node analysis was carried out with PCR analysis. If it it absolutely was reported as macrometastases, axillary lymphatic tissue clearance was performed. Intra-operative radiography of the excised specimens confirmed the presence of the lesion and determined that the lesion was clear of the margins. Final pathology results were discussed at the multi-disciplinary meeting. Patients who underwent a standard WLE were assessed for their suitability for a satisfactory cosmetic outcome. Therefore, in this procedure, no glandular mobilization was performed. According to the NICE guidelines, our criteria for margin clearance was 1mm for an invasive cancer and two mm for DCIS.

 

Results:

The procedures employed were the tennis racquet technique for upper outer quadrant tumors (n= 19); vertical scar techniques (n=15), Z-plasty techniques (n=13), Grisotti flaps (n=3), intramammary flaps  (n=2), B-plasty (n=1) and reduction mammoplasty (n=1). There was a significant difference in the age of the patients (p=0.0003). Our results showed that OPS favored younger patients. Both groups showed a majority of ductal carcinomas, with lobular following second in the OPS group and DCIS in the WLE group. There was no significant difference between both groups in terms of the type (p value=0.2) and grade of the tumor . OPS excised a significantly larger tumor size compared to the WLE procedure (p<0.05). Significantly heavier specimens were removed from patients undergoing OPS relative to WLE (P<0.05) which is illustrated. The mean margin clearance for the OPS group was 4.7 mm. As for the WLE group, it was 3.5 mm. There was no statistical difference between the margin clearances of both of the groups (p value=0.10). Of the specimens with involved margins, 14 re-excisions and 5 completion mastectomies were performed in the WLE group. A further two completion mastectomies were due to one patient being identified as later having the BRCA1 mutation and another developing complications related to radiotherapy. In the OPS group there was two wider excisions and six completion mastectomies. Out of the six completion mastectomies in the latter group, three of these patients opted for a reconstruction. Two underwent implant based reconstructions, and one underwent a deep inferior epi gastric flap reconstruction. Margin involvement in the OPS group primarily affected those patients with lobular tumors or DCIS extending over 40 mm in size (n=6), while patients with involved margins in the WLE

group had ductal carcinoma or in situ disease.

Discussion:

We found that OPS was being performed on significantly larger tumors than those treated with a WLE (32.1 mm vs. 18.8 mm; p<0.05). This was similarly the case where the weight of the tumor was concerned (177 grams vs. 73.1 grams; p<0.05). Hence, some patients underwent a quadrantectomy which is not a routine practice in the UK. These procedures resulted in a decrease in the number of mastectomies we would have performed in these circumstances. The findings from our study are in agreement with the existing literature on operative outcomes in OPS surgery. Clough [15] and Down et al. have previously reported a preference for OPS in managing larger tumors. An average tumor size of 32 mm in a series of 101 therapeutic mammoplasties was reported by the former study, while the latter reported an average size of 23.9 mm in 37 OPS patients. Similarly, previous studies have reported specimen weights for therapeutic mammoplasty ranging from 222-236

Conclusion :

OPS was performed in patients with larger tumors suggesting it should be considered. Our study has demonstrated acceptable cosmetic outcomes and low recurrence rates.

Bottom line: This work is partly presented at 3rd World Conference on Breast and Cervical Cancer September 24-25, 2018 Abu Dhabi, UAE

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