Slanic Prahova, 20 Septembrie 2012

Constantin Munteanu Institutul National de Recuperare, Medicina Fizica si Balneoclimatologie

Asociatia Romana de Balneologie

Cercetarea stiintifica a factorilor naturali terapeutici

Institutul Naţional de Recuperare, Medicină Fizică şi Balneoclimatologie

Laborator Culturi Celulare

SCURT ISTORIC AL BALNEOLOGIEI ROMANESTI

Institutul National de Recuperare, Medicinã Fizicã si Balneoclimatologie a fost infiintat in anul 1924 sub denumirea de "Institut de Balneologie", prin stradania sustinuta a unor personalitati marcante ale timpului - medicale si nemedicale: din partea Ministerului Sanatatii, a Crucii Rosii Romane, a Patriarhiei, a Ministerului Apararii Nationale si alte institutii.

De mentionat ca la acea vreme, a fost al doilea institut de profil din Europa (dupa cel din Franta).

UTILIZAREA MEDICALA A FACTORILOR NATURALI TERAPEUTICI

Specialistii în recuperare, medicina fizica si balneologie intervin prin mijloace terapeutice specifice în tratamentul urmatoarelor afectiuni generatoare de deficiente functionale:

a) afectiuni posttraumatice;b) afectiuni musculo-scheletale;c) afectiuni reumatismale inflamatorii;d) afectiuni cardiovasculare;e) afectiuni respiratorii;f) afectiuni ginecologice;g) afectiuni dermatologice; h) afectiuni neurologice; i) afectiuni neuropsihomotorii;

STUDII GEOLOGICE SI HIDROLOGICE ASUPRA RESURSELOR NATURALE TERAPEUTICE

Cercetarile geologice, indiferent de metodele folosite deductive sau inductive, se finalizeaza prin materiale de sinteza (studii, rapoarte, etc.) însoţite de materiale cartografice. În baza concluziilor acestor materiale se procedeaza la detalierea cunoaşterii în vederea valorificarii economice ale unor resurse minerale terapeutice.

STUDII BIOCLIMATICEProcedura de lucru în cazul bioclimatului (în absenţa

momentană a unei reglementări unitare la nivel european) se referă la prelucrări (calcule) de specialitate ale datelor climatice şi microclimatice, care să conducă la estimarea valorică a unor indici bioclimatici. Dintre aceşti indici se menţionează:

- indicele de confort termic (bazat pe temperatura efectiv echivalentă, stabilită cu ajutorul temperaturii şi umidităţii aerului, precum şi a intensităţii vântului);

- indicele de stres climatic cutanat (referitor la senzaţia de cald/frig resimţătă de de organismul uman, în funcţie de temperatura aerului şi intensitatea vântului);

- indicele de stres climatic pulmonar (bazat pe senzaţia de hidratare/deshidratare a mucoasei pulmonare, în funcţie de tensiunea vaporilor de apă din aerul atmosferic);

- indicele de stres bioclimatic total (însumarea valorilor celor două stresuri climatice anterioare).

La aceşti indici se pot adăuga şi valorile altor indici bioclimatici, în măsura în care se pot obţine datele necesare: indici de aeroionizare, etc

ANALIZA FIZICO-CHIMICA A FACTORILOR NATURALI TERAPEUTICI

Calitatea factorilor naturali este determinată efectiv de proprietãţile lor fizico-chimice rezultate din compoziţia de substanţe minerale sau organice, a gazelor dizolvate sau de particulele aflate în suspensie şi organismele vii prezente.

ANALIZA MICROBIOLOGICA A FACTORILOR NATURALI TERAPEUTICI

Analiza microbiologica a diferitelor probe se realizeaza în scop preventiv, de diagnostic, prognostic sau terapeutic.

Biologul care lucreaza într-un laborator de microbiologie are responsabilitatea prelevarii corecte a probelor, executarii analizei de laborator a probei, validarea rezultatelor si la nevoie confruntarea acestora cu date complementare (clinice, epidemiologice etc.), participând deci în mod indirect la interpretarea rezultatelor obtinute.

CERCETARI FIZIOLOGICE SI FARMACODINAMICE

Cunoasterea problemelor generale privind efectele factorilor naturali, locul şi mecanismul lor de acţiune, are o importanţă fundamentalăpentru o terapeutică raţională.

Activitatea biologică a factorilor naturalipresupune o interacţiune iniţială, de ordin chimic sau fizico-chimic, cu moleculele componente ale materiei vii. Această acţiune primară, la nivel molecular, declanşează un complex de reacţii, avâd drept rezultat efectul farmacologic global, răspunsul organismului la medicamente.

Interacţiunea la nivel molecular, de ordin fizico-chimic, biochimic - se numeste acţiune farmacodinamică. Efectul farmacodinamic reprezintă rezultatul complexului de reacţii declanşate în organism de acţiunea primară. Un medicament nu creează funcţii noi în organism, ci numai accelerează sau diminuă procesele şi fenomenele fiziologice dereglate.

CERCETARI AVANSATE LA NIVEL CELULAR SI MOLECULAR

Prin studii in vitro se pot urmãri: morfologia celularã, sinteza proteicã, secreţia anumitor substanţe, metabolismul celular, interacţia celulelor prin receptori celulari cu diferiţi liganzi, captarea sau eliberarea electroliţilor ori a altor tipuri de substanţe care ajung în mediul celular.

Evaluarea modificărilor la nivel celular şi molecular se poate realiza prin studii de microscopie optică, în care este urmãritãmorfologia celulara, studii de viabilitate celulara, studii imunocitochimie, studii proteomice realizate prin tehnici specifice, incluzând electroforeza şi Western Blottingul, determinarea parametrilor biochimici de baza la nivelul mediului de culturã, studii asupra fiziologiei celulare, studii privind senescenţa celularã, studii privind semnalizarea celularã.

PIP2PIDAG

MUNC

PLC

DshAkt/PKB

PKC

Ligand A

Li

IP3

+

IP2IP

inositol

Lithium-ResponsiveGene Network

Long-term LithiumEffects

Ca2+

Li+

APC

GSK-3β

AXIN

PP2A

GBP

β-catenin

Regulation of PKCresponsive genes

TCF/LEF responsivegenes

TCF/LEF

IMPase

IMPase

CMP-PA FrizzledPI-3K

αγ β

β-catenin

β-catenin

Phosphorylation, Translocation oftranscription factors

NEUROPROTECTIONNEUROPLASTICITYSYNAPTIC FUNCTION

+

+

+

+-

- -

- --

P

Protein degradation

Wnt

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expresiei GSK-3β în culturile gliale de 14 zile

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CERCETARI AVANSATE LA NIVEL CELULAR SI MOLECULAR

CERCETARI AVANSATE LA NIVEL CELULAR SI MOLECULAR

AbstractObjective: Exploring the speleotherapy effects on

morphology and physiology of dermal and pulmonary fibroblast obtained from Wistar rats tissue in normal conditions and after induction of experimental “astma”awareness with ovalbumin.

Exploration of the speleotherapeutic potential through the cellular and molecular biology techniques

Munteanu C., Munteanu D., Simionca I., Cinteza D., Hoteteu M.;

Materials and methods:

Dermal and pulmonary fibroblast cultures were initiated by enzymatic techniques from appropriate tissue taken of each group Wistar rats. Morphological monitoring was done by phase contast microscopy; biochemical andmolecular changes of cultures obtained from animals treated speleothropic compared to control, was experimental establised by electrophoresis and Western Blotting techniques.

Results: Experimental data revealed the expression of several proteins after the speleotherapeutic treatment. These data were analysed compared with control, using a specific software.

Conclusions: Speleotherapeutic treatment of Wistar rats caused significant differences in morphology and protein expression of dermal and pulmonary fibroblatst grown in the laboratory. These differences support the protective effects of speleotherapy compared with data obtained from animals untreated and sensitized with ovalbumin, having induced experimental asthma status.

38th World Congress of ISMH, the International Scientific Meeting of Medical Hydrology Balneology and Climatology, Lanjarón-Granada Spain, 20-23 June 2012

Health effects attributed to endocrine disrupting compounds fromHealth effects attributed to endocrine disrupting compounds from mud and lake waters sourcesmud and lake waters sourcesConstantin MunteanuConstantin Munteanu 1,21,2, Delia Cinteza, Delia Cinteza 1,21,2, Diana Munteanu, Diana Munteanu1,21,2, Mihai Hoteteu, Mihai Hoteteu11, Horia L, Horia Lããzzããrescurescu11, Daniela Poenaru, Daniela Poenaru1,21,2

11 National Institute of Rehabilitation, Physical Medicine and BalNational Institute of Rehabilitation, Physical Medicine and Balneoclimatology, Bucharest, Romanianeoclimatology, Bucharest, Romania22 Romanian Association of Balneology ( Romanian Association of Balneology ( www.bioclima.ro )

EE--mail: mail: secretar@bioclima.ro

Artemia salina (L.) is a primitive aquatic arthropod (salt lakes) of the Artemiidae family (figure 3) with an age of about 100 million years. Linný (1758) described it as Cýncer salinus but 61 years later, Leach (1819) transferred it to Artemia salina. It was reported for the first time in Urmia Lake in 982 by a Iranian geographer (Asem, 2008).

Clinical data suggest that peloidotherapy is useful in preparing the uterus for in vitro fertilization, but without scientific evidence. Mud and water from Bear Lake must be analyzed, both in terms of physico-chemical, microbiological and in terms of phyto-and zooplankton to detect organisms that grow in the lake and are structural and biological determinants of sludge role in studies on infertility. Previous studies conducted at the Institute of Balneology, Bucharest, have suggested the existence in the lake water of certain substances having similar effects on theendometrium as the female sexual hormones.

Salt water and mud from lake Ursu heliothermal contain hormones released by Artemia salina: human estrogen-like SU 95%, respectively human progesterone-like 0.7 to 0.8 mg% (Stoicescu Munteanu, 1977). They are used to treat gynecological diseases: ovarian failure, infertility, etc.

Action: Infertility (sterility) is a health problem affecting 15% of couples of reproductive age. Today only a few are known about causes and treatment options involved in the pathology of infertility, while a number of issues remain unknown. Sapropelic mud and salt water have a high content of salt and human-like estrogen and progesterone hormones (liberated by Artemia salina arthropode). High temperature of mud and salt water increases local circulation, implicitly the absorbtion of salt and hormones. Salt decreases local inflammation, heals genital lesionsand dissolves adherences. Hormones, with almost the same chemical structure as the human ones, are received by ovaria, hipophysis and hypothalamus as own, effectively stimulating and balancing the own hormonal sinthesis. Ovarian synthesis of estrogen and progesterone increases, regulating menstruations, optimising vaginal pH and ovulation and prepare uterus for egg implantation.

Endocrine disruptors are estrogen-like and anti-androgenic chemicals in the environment. They mimic natural hormones, inhibit the action of hormones, or alter the normal regulatory function of the endocrine system and have potential hazardous effects on male reproductive axis causing infertility.

Although testicular and prostate cancers, abnormal sexual development, undescended testis, chronic inflammation, Sertoli-cell-only pattern, hypospadias, altered pituitary and thyroid gland functions are also observed, the available data are insufficient to deduce worldwide conclusions.

Health effects attributed to endocrine disrupting compounds include a range of reproductive problems (reduced fertility, male and female reproductive tract abnormalities, and skewed male/female sex ratios, loss of fetus, menstrual problems); changes in hormone levels; early puberty; brain and behavior problems; impaired immune functions; and various cancers.

The Endocrine Society released a scientific statement outlining mechanisms and effects of endocrine disruptors on “male and female reproduction, breast development and cancer, prostate cancer, neuroendocrinology, thyroid, metabolism and obesity, and cardiovascular endocrinology,” and showing how experimental and epidemiological studies converge with human clinical observations “to implicate EDCs as a significant concern to public health.” The statement noted that it is difficult to show that endocrinedisruptors cause human diseases, and it recommended that the precautionary principle should be followed.

INTRODUCTION

Endocrine disruptors are substances that "interfere with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body that are responsible for development, behavior, fertility, and maintenance of homeostasis (normal cell metabolism)." They are sometimes also referred to as hormonally active agents, endocrine disrupting chemicals, or endocrine disrupting compounds (EDCs).

EDC studies have shown that endocrine disruptors can cause adverse biological effects in animals, and low-level exposures also cause similar effects in human beings. The term endocrine disruptor is often used as synonym for xenohormone although the later can mean any naturally occurring or artificially produced compound showing hormone-like properties (usually binding to certain hormonal receptors). The term endocrine disruptor was coined at the Wingspread Conference Centre in Wisconsin, in 1991. One of the early papers on the phenomenon was by Theo Colborn in 1993.

Figure 3 Artemia salina (L.)

The coronary heart disease (CHD) produces 1/5 from all deaths. Premenopausalwomen have a low incidence of coronary heart disease, which rises rapidly after the menopause towards incidence levels observed in men (1, 7). Many studies show that estradiol reduces ischemia / reperfusion lesions (5, 6, 8, 9, 11). The estradiol effects on cardiovascular system are multiple and the action mechanisms are not completely understood. The aim of the present study was to characterize the effect of 17β-estradiol on cardiomyocytes contractile function under oxidative stress conditions.

RESULTS AND DISCUSSIONS

Structural Changes Following H2O2 Treatment - The oxidative stress produces α-actin immunohistochemical reaction alteration proportionally with the H2O2 concentration (Fig. 1-3). On 50 µM H2O2 concentration no reaction was observed. Following estradioltreatment, α-actin was detected in all of three experiments (Fig. 4-7). α-tropomyosin was detected in cardiomyocytes with oxidative stress at all H2O2 concentrations with analterated morphology (Fig. 9-11). The estradiol treatment partly counteracts the effects of oxidative stress.

Cardiomyocytes contraction rate - The 10 µM H2O2 induces a decrease with 34% of contraction number while at the 25 and 50 µM H2O2 no contractions were observed. Estradiol treatment produces a decrease with 16% of contraction number and no contractions were observed at 25 and 50 µM H2O2 .

Na+/K+-ATP-ase Activity - Oxidative stress induces a significant decrease of Na+/K+-ATP-ase activity proportionally with H2O2 concentration. Estradiol treatment partly protects the Na+/K+-ATP-ase activity.

CONCLUSIONS

Oxidative stress is associated with muscle protein modifications. At high doses of H2O2, actin and tropomyosin filaments aspect is dramatically changed.

The alteration of cardiomyocytes contractility is a complex process that involves changes of proteins activity at multiple cellular levels. Estradiol treatment attenuates the effects of H2O2 upon muscle proteins.

MATERIALS AND METHODS

Primary cultures of neonatal rat cardiomyocytes were prepared as previously described (12). Briefly, 1- to 2-day old Wistar rats were euthanized, hearts excised and ventricles minced in phosphate- buffered saline solution. Serial digestions with 0.125% trypsin were performed at 370C; cell pellets were resuspended in Dulbecco’s Modified Eagle’s Medium Nutrient Mixture F-12 HAM (DMEM-F12) with 10% fetal calf serum, 100 UI/ml penicillin, 100 µg/ml streptomycin.

Estradiol Treatment - The 3 days cells cultures were treated with 125 pg/ml 17β-estradiol for 48h. H2O2 Treatment - Cells were treated with 10, 25 and 50 µM H2O2 by replacing the culture medium with serum-free medium containing H2O2.

Structural Changes Following H2O2 Treatment

Immunohistochemical staining was performed using mouse monoclonal anti α-sarcomeric actin and monoclonal anti-sarcomeric tropomyosin antibodies. Cardiomyocytes contraction rate -The mean contraction rate of 10 myocytes / culture well was determined microscopically.

Na+/K+-ATP-ase Activity - ATP-hydrolysis was measured at 37°C following the method of Garner (3). Na/K-ATPase –dependent ATP hydrolysis is reported as nmoles Pi released/h/mg protein.

Fig. 1 – Positive immunohistochemicalreaction for α-actin; x400.

Fig. 2 -Neuniformly immunostaining for α-actin after 10 µM H2O2; x400.

Fig. 3 – Reduced immunostaining for α-actin after 25 µM H2O2; x400.

Fig. 5 - Positive immunohistochemical reaction for actin after treatment with estradiol - 10 µM

H2O2 ; x400.

Fig. 6 -Immunohistochemical reaction for actinafter treatment with estradiol - 25 µM H2O2 ;

x400.

Fig. 7 – Alterated distribution of α-actin after treatment with 50 µM H2O2 and estradiol;

x400.

Fig. 8 -Positive immunohistochemical reaction for α-tropomyosin; x400.

Fig. 9 - Positive immunohistochemical reaction for α-tropomyosin after treatment with 10 µM

H2O2 ; x400.

Fig. 10 - Positive immunohistochemical reaction for α-tropomyosin after treatment with 25 µM H2O2 ;

x400.

Fig. 11 -Alterated distribution of α-tropomyosinafter treatment with 50 µM H2O2 ; x400.

Fig. 13 - Positive immunohistochemical reaction for α-tropomyosin after treatment with estradiol

- 10 µM H2O2 ; x400.

Fig. 14 -Immunohistochemical reaction for α-tropomyosin after treatment with estradiol - 25

µM H2O2 ; x400.

Fig. 15 -Modified distribution of α-tropomyosinafter treatment with 50 µM H2O2 ; x400.

Fig. 4 - Positive immunohistochemical reaction for α-actin after estradiol treatment; x400.

Fig. 12 - Positive immunohistochemical reaction for α-tropomyosin after estradiol treatment; x400.

88thth FENS Forum of NeuroscienceFENS Forum of Neuroscience, Barcelona, Spain, July 14, Barcelona, Spain, July 14--18 2012 18 2012 Presentation Code: 127.18 Abstract Number: 186 Poster Board NumbPresentation Code: 127.18 Abstract Number: 186 Poster Board Number: C117 er: C117

Munteanu C.1, Munteanu D.1, Cinteza D.21Physiology, Cell Cultures, National Institute of Rehabilitation, Physical Medicine and Balneoclimatology, Romania

2 Research and Medical Department, National Institute of Rehabilitation, Physical Medicine and Balneoclimatology, Romania

INTRODUCTIONMany authors proposed that

lithium is a good treatment for migraine and bipolar disorders. We intend to use lithium mineral waters, defined as natural therapeutic factors, with lithium at safe concentrations, in prophylaxis of anxiety and bipolar depression.

One important question that arises for any psychiatrist is if the lithium content of the mineral water can or not has a therapeutic or a prophylactic value. An initial answer is that preliminary studies on glial cell cultures showed a significant modification in cell biochemistry and morphology. These modifications constitute an argument for us to go on with future experiments to clarify this issue. Secondly, if we look to the concentration values we have to remark that one liter of water contains 1.1 mEq of lithium.

This concentration is important for us, in our intention to reach progressively the therapeutic index of the lithium ions.

GFAP is a very sensitive and specific marker for rapid astrocytic response to injury and disease. Increase of GFAP in astrocytes occurs gradually throughout the adult lifespan. Since GFAP normally increases with age and there is a wide variation in the collection and processing of human brain tissue, it is difficult to demonstrate mild gliosis by immunocytochemistry. In control and 1 mM LiCl glial cells cultures, immunodetection of GFAP shows a weak reaction. 2mM LiCl determine an intensification of GFAP expression.

MATERIALS AND METHODSThe primary glial cells cultures

were initiated from the brain of Wistar rat pups aged 1-3 days, in keeping with known techniques. After the meninx is removed, the brain is passed through a 60µm nytex and the cells thus obtained are directly plated on glass Petri dishes with a 60 mm diameter.

The cells have been grown in a DMEM medium (Sigma), with 4500 mg/l of glucose, 25 mM HEPES, 100-u/ml penicillin and 100 µg/ml streptomycin (Sigma). The medium was supplemented with 15% calf fetal serum (Gibco). The medium was first replaced after 24 hrs and then every three days. The primary cells cultures will be maintained in a humidified atmosphere, at 370C, with 5% CO2.

Immunohistochemical detections of GSK-3βwere made by an indirect immunoperoxidase method. GSK-3β plays a critical role in the central nervous system by regulating various cytoskeletal processes as well as long-term nuclear events, its inhibition may underlie some of the long-term therapeutic effects of mood-stabilising agents. The results presented here suggest that at 2 mM LiCl the inhibition of GSK-3β expression is much accentuated and this correspond to the majority of papers which indicate GSK-3β as main target of lithium action.

The initial primary cultures most likely contained neurons, astrocytes, oligodendrocytes, other glial cells, endothelial cells and undifferentiated cells. The shaking method of separating cells allowed for the selection of astrocytes, which are more readily adherent.

The inositol polyphosphate phosphatasesselectively remove phosphate groups from various phosphatidylinositols, which generate second messengers in response to extracellular signals. SHIP regulates intracellular calcium concentrations and may also modulate the Rad signalling pathway.

Our results reflect a cortical reaction for SHIP in the control glial cultures, but the distribution of SHIP proteins becomes heterogenic in the case of lithium treatment with 2mM LiCl.

PIP2PIDAG

MUNC

PLC

DshAkt/PKB

PKC

Ligand A

Li

IP3

+

IP2IP

inositol

Lithium-ResponsiveGene Network

Long-term LithiumEffects

Ca2+

Li+

AP

C

GSK-3β

AX

IN

PP2A

GBP

β-catenin

Regulation of PKCresponsive genes

TCF/LEF responsivegenes

TCF/LEF

IMPase

IMPase

CMP-PA FrizzledPI-3K

αγ β

β-catenin

β-catenin

Phosphorylation, Translocation oftranscription factors

NEUROPROTECTIONNEUROPLASTICITYSYNAPTIC FUNCTION

+

+

+

+-

- -

- --

P

Protein degradation

Wnt

Adapted after Lenox and all, 2003

MORPHOLOGY

E-mail: secretar@bioclima.ro

Romanian Association of Balneology ( www.bioclima.ro )

LiCl 1 mM

LiCl 1 mMCONTROL

MORPHOLOGY

GSK – 3ß

GFAP

SHIP

LITIU 1mMCONTROL LITIU 2mM MARIA Water

METHODS

LITHIUM LITHIUM –– TRACE ELEMENT WITH NEUROLOGICAL EFFECTS IN AFECTIVE DISORDERSTRACE ELEMENT WITH NEUROLOGICAL EFFECTS IN AFECTIVE DISORDERS

Although the cultures were determined to be highly pure, the astrocytes presented showed a great variation in morphology. One reason for this variety could be the aphysiologic conditions under which the cells were grown. Normally, cells in the brain grow at an oxygen concentration of about 2%. Atmospheric oxygen concentrations are usually 20%; at high oxygen concentration, many cells in the body have been shown to exhibit unusual morphologies.

STUDII CLINICE ASUPRA EFICIENŢEI TERAPEUTICE A FACTORILOR NATURALI

Selectia si recrutarea unor participanţi la studiu adecvaţi – timpul de consultare are şanse sensibile sa fie mai lung decât în situaţii normale.

Obţinerea consimţământului informat de la toţi participanţii la studiu. Conceptul de obţinere a consimţământului informat este considerat principiul de bază al GCP.

Realizarea modelului curativ balnear integrat are loc in baza cercetarilor experimentale şi clinice desfasurate, avand astfel o fundamentare stiintifica, biologica si medicala actualizata, obtinuta prin tehnici experimentale moderne şi studii clinice.

ROLUL CERCETARII ŞTIINŢIFICE ÎN PROMOVAREA SERVICIILOR BALNEARE

Pentru medicina balnearã, importanţa cercetãrii fundamentale echivaleazã cu promovarea unei staţiuni balneare prin cunoaşterea factorilor naturali cu potenţial terapeutic ce stau la baza activitãţii medicale, identificarea mecanismelor biologice de nivel celular şi molecular prin care factorii naturali actioneazã asupra organismului şi a modalitãţilor de rãspuns fiziologic pe care organismul le poate utiliza în încercarea de a se adapta la condiţia stimulãrii prin factorul terapeutic.

O valorificare strategică a potenţialului turismului balnear românesc va permite repoziţionarea acestuia pe piaţa internă şi internaţională. Reuşita acestei acţiuni depinde de implicarea hotărâtă a factorilor de decizie de la nivel macro şi microeconomic si de utilizarea cercetarii stiintifice in promovarea turismului balneo-climatic.

Va multumesc Va multumesc !!

Dr. Biol. Constantin MunteanuDr. Biol. Constantin Munteanuwww.bioclima.ro

Email: secretar@bioclima.ro

Institutul Naţional de Recuperare, Medicină Fizică şi Balneoclimatologie