This study compared different methods for sampling gingival crevicular fluid (GCF) in patients with severe chronic periodontitis. The methods compared were paper strips, paper points, and washing techniques. The goal was to identify a method that could detect both microbiota and immunologic markers like cytokines. The results showed that paper strips detected cytokines like IL-6 and IL-8 more often than paper points. Additionally, washing techniques detected higher levels of proteases from Porphyromonas gingivalis compared to paper strips or points. Therefore, the study concluded that different sampling methods are better suited for detecting certain parameters, and a combination may be needed to analyze both microbiota and inflammatory markers in GCF.
The future of dentistry and periodontics lies in regeneration. The goals of periodontal therapy lies in not only the arrest of periodontal disease progression but also regeneration of the lost periodontal structures. This presentation provides a review of the current understanding of the regeneration of the periodontium and the procedures involved to restore the periodontal tissues around the teeth.
Furcation involvement is a common sequela of severe chronic periodontal disease. Its effective management has a profound influence on the outcome of periodontal therapy.
The defense mechanism of gingiva includes GCF, Saliva, epithelial barrier and connective tissue cells. All these protect the periodontium from bacterial invasion.
The future of dentistry and periodontics lies in regeneration. The goals of periodontal therapy lies in not only the arrest of periodontal disease progression but also regeneration of the lost periodontal structures. This presentation provides a review of the current understanding of the regeneration of the periodontium and the procedures involved to restore the periodontal tissues around the teeth.
Furcation involvement is a common sequela of severe chronic periodontal disease. Its effective management has a profound influence on the outcome of periodontal therapy.
The defense mechanism of gingiva includes GCF, Saliva, epithelial barrier and connective tissue cells. All these protect the periodontium from bacterial invasion.
Oral submucous fibrosis is defined as “An insidious chronic disease affecting any part of the oral cavity and sometimes the pharynx. A wide range of treatment including drug therapy, surgical therapy, and physiotherapy have been attempted till date, with varying degrees of benefit, but none have been able to cure this disease.
The treatment depends on the level of clinical involvement. At a very early stage, cessation of the habit is adequate. Medical/surgical treatment is necessary for moderate to severe cases. Surgical treatment is the method of choice in patients with marked limitation of mouth opening or in patients not responding to the conservative management.
Epidemiology of gingival & periodontal diseasesChetan Basnet
It is the “study of the distribution and determinants of health related states or events in a specified population, and the application of this study to control of health problems.”
-John M. Last(1988)
THIS PRESENTATION INCLUDES:
INTRODUCTION
MAIN BLOOD SUPPLY BRANCHES TO PERIODONTIUM
BLOOD SUPPLY TO MAXILLARY TEETH AND PERIODONTIUM
BLOOD SUPPLY TO MANDIBULAR TEETH AND PERIODONTIUM
VENOUS DRAINAGE OF MAXILLARY AND MANDIBULAR TEETH AND PERIODONTIUM
BLOOD SUPPLY TO EACH COMPONENT OF PERIODONTIUM
CLINICAL SIGNIFICANCE OF BLOOD SUPPLYING THE PERIODONTIUM
CLINICAL CORELATIONS WITH GINGIVITIS AND PERIODONTITIS
CONCLUSION
REFERENCES
TMJ is a ginglymo-diarthroidal joint that is freely mobile with superior and inferior joint spaces separated by articular disc.
The type of imaging technique depends upon the clinical problems associated, so either imaging of hard tissue (OSSEOUS) or soft tissue is desired.
Certain protocols are to be taken care before the imaging procedure:
the amount of diagnostic information available from particular imaging modality.
The cost of examination
The radiation dose
Aberrant Frenum !!
No worries... When Frenectomy is here.
Hello Periodontists,
Here's the entire process of Frenectomy in a nutshell and various ways to encounter the same.
Lets Shoot ...
This presentation gives the brief idea of the various guidelines carried out to study the genetic damage to cells when there is a discover of new active molecule.
Oral submucous fibrosis is defined as “An insidious chronic disease affecting any part of the oral cavity and sometimes the pharynx. A wide range of treatment including drug therapy, surgical therapy, and physiotherapy have been attempted till date, with varying degrees of benefit, but none have been able to cure this disease.
The treatment depends on the level of clinical involvement. At a very early stage, cessation of the habit is adequate. Medical/surgical treatment is necessary for moderate to severe cases. Surgical treatment is the method of choice in patients with marked limitation of mouth opening or in patients not responding to the conservative management.
Epidemiology of gingival & periodontal diseasesChetan Basnet
It is the “study of the distribution and determinants of health related states or events in a specified population, and the application of this study to control of health problems.”
-John M. Last(1988)
THIS PRESENTATION INCLUDES:
INTRODUCTION
MAIN BLOOD SUPPLY BRANCHES TO PERIODONTIUM
BLOOD SUPPLY TO MAXILLARY TEETH AND PERIODONTIUM
BLOOD SUPPLY TO MANDIBULAR TEETH AND PERIODONTIUM
VENOUS DRAINAGE OF MAXILLARY AND MANDIBULAR TEETH AND PERIODONTIUM
BLOOD SUPPLY TO EACH COMPONENT OF PERIODONTIUM
CLINICAL SIGNIFICANCE OF BLOOD SUPPLYING THE PERIODONTIUM
CLINICAL CORELATIONS WITH GINGIVITIS AND PERIODONTITIS
CONCLUSION
REFERENCES
TMJ is a ginglymo-diarthroidal joint that is freely mobile with superior and inferior joint spaces separated by articular disc.
The type of imaging technique depends upon the clinical problems associated, so either imaging of hard tissue (OSSEOUS) or soft tissue is desired.
Certain protocols are to be taken care before the imaging procedure:
the amount of diagnostic information available from particular imaging modality.
The cost of examination
The radiation dose
Aberrant Frenum !!
No worries... When Frenectomy is here.
Hello Periodontists,
Here's the entire process of Frenectomy in a nutshell and various ways to encounter the same.
Lets Shoot ...
This presentation gives the brief idea of the various guidelines carried out to study the genetic damage to cells when there is a discover of new active molecule.
Induction of transformation by a deoxyribonucleic acid fraction isolated from...Babita Neupane
This is a highlight of the one of the groundbreaking paper of early 1940's in field of molecular biology.
Induction of transformation by a deoxyribonucleic acid fraction isolated from Pneumococcus Type III. Avery, O.T., Macleod, C. M., McCarty, M. (1944) The journal of experimental medicine 79(2):137-58.
Osseointegration, definition, history, process of osseointegration, factors influencing osseointegration, methods for evaluation of osseointegration, failure of osseointegration
Definition of periodontal pocket, classification, Histopathology of periodontal pocket, microflora involved, pathogenesis, periodontal pocket as a healing lesion, microtopography of root surface, treatment of periodontal pocket
Smoking and periodontal disease, smoking as a risk factor, incidence of smoking, effects of smoking on periodontium, smoking and gingivitis and smoking and periodontitis, effect of surgical and non surgical therapy on smokers
Systemic Peridoontology, link between systemic health and periodontology, diabetes and periodontology, Pregnancy and Peridotology,Nutrition and periodontology
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Acute scrotum is a general term referring to an emergency condition affecting the contents or the wall of the scrotum.
There are a number of conditions that present acutely, predominantly with pain and/or swelling
A careful and detailed history and examination, and in some cases, investigations allow differentiation between these diagnoses. A prompt diagnosis is essential as the patient may require urgent surgical intervention
Testicular torsion refers to twisting of the spermatic cord, causing ischaemia of the testicle.
Testicular torsion results from inadequate fixation of the testis to the tunica vaginalis producing ischemia from reduced arterial inflow and venous outflow obstruction.
The prevalence of testicular torsion in adult patients hospitalized with acute scrotal pain is approximately 25 to 50 percent
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
1. COMPARISON OF GINGIVAL
CREVICULAR FLUID SAMPLING
METHODS IN PATIENTS WITH SEVERE
CHRONIC PERIODONTITIS
Arndt Guentsh, Martin
Kramesberger, Aneta Sroka,
Wolfgang Pfister, Jan Potempa, and
Sigrun Eick
J Periodontol, July 2011
Presented By:
Pallavi Prashar
2. Introduction
• In periodontal disease, gingival crevicular fluid
(GCF) is an inflammatory exudate.
• GCF contains substances from the host and
supra- and subgingival bacteria.
• Host constituent include
▫ molecule from blood and periodontal tissues
▫ inflammatory and immune cells that infiltrate into
periodontal tissue are found in GCF together with
markers of inflammation, including
Enzymes
Cytokines
Interleukins (Ils)
• Products of tissue breakdown.
3. • The analysis of GCF and subgingival microflora has
become more important in diagnosis and therapy of
periodontal diseases.
• The presence of large number of periodontopathic
bacteria in GCF, such as Actinobacillus
actinomycetemcomitan (AAC) and member of red
complex organism including P.gingivalis, T.forsythia,
T.denticola indicates clinically important microbial
infection.
• Arginine-specific gingipains (Rgps) are an important
virulence factors of P.gingivalis that plays major role in
maintenance of inflammatory conditions in
periodontitis. They are able to impair neutrophil
function and degrade the extracellular matrix and
bioactive peptides such as complement factor-C5,
prekallikerin, and kininogens. Furthermore Rgps can
inactivate IL-6 and inhibitor of neutrophil proteases.
4. • In periodontitis there are increased levels of
expression and synthesis of IL-1, TNF-alpha,
and IL-6 and 8 in periodontal tissue.
• An effective host response to bacteria is
primarily mediated by neutrophil and
characterized by influx of neutrophil into
gingival crevices.
• Elastase levels are among the highest of any
proteinase activity determined in GCF during
periodontal inflamation. The assessment of the
granulocyte elastase activity in GCF can serve as
marker of intracrevicular granulocyte activity.
5. Different techniques have been employed for sampling
the content of periodontal pocket.
• Subgingival bacteria can be sampled with curettes or
paper points, whereas cytokines and host enzymes
were usually collected with filter paper strips.
• There are considerable variations in the application of
the paper-strip method of collection which can be
intracrevicular (strip is inserted unless resistance is
felt) or extracrevicular (strip kept at enterance of
crevice).
• Washing technique is an possible alternative when
other sampling method fails.
6. Aim of the study
• To identify a method that could be used for
different purposes (e.g., microbiota and
immunologic variables)
• Moreover, different sampling techniques were
tested to effectively detect a parameters of
special interest, in this case Rgps.
• Because of missing data in literature , an
additional aim of this study was to investigate
the level of Rgps and its correlation to
P.gingivalis in GCF.
7. Materials and Methods
• Subject recruitment
▫ Thirty six subjects aged 38 to 56 years with chronic periodontitis
were recruited.
▫ The definition of chronic periodontitis was based on the
classification system of the International Workshop for
Classification System of Periodontal Diseases and Conditions from
1999.
▫ Patients with generalized chronic periodontitis were included if
they were >35 years of age and demonstrated attachment loss of
>5 mm at >30% of sites.
▫ After the hygiene phase plaque was <35%.
• To ensure similar periodontal conditions for comparison of
sampling methods, each molar per quadrant had a site with
probing deapth between 5 and 7 mm.
• Subjects with significant disease (e.g., diabetes melluitus,
cancer, or coronary heart disease), antibiotic therapy within the
past 6 months, and females who were pregnant and lactating
were excluded.
• Only non-smokers with no history of smoking were included.
8. Clinical Assessment
• Probing depths were measured with UNC 15 probe at six sites
per tooth.
SAMPLE COLLECTION
• Patients were randomized per lot into one of the three
groups.
▫ In Group 1 paper strips were compared to paper points.
▫ In Group 2 paper strips were compared to washing techniques.
▫ In Group 3 paper points were compared with washing
techniques.
• GCF was sampled in each patient using two sampling
techniques on only molars with probing depth from 5 to
7mm.
• One method was performed at upper and lower molars on
right side and the other method was performed at
corresponding molar on left side of oral cavity.
• After 1 week, the collection of samples were repeated using
the opposite sites.
• Samples were collected in the morning 2 to 3 hours after
breakfast.
9. • The sites to be sampled were isolated with cotton rolls and
gently air dried.
• Paper strips and paper points were gently placed for 30 seconds
into pocket until minimum resistance were felt.
• Samples were eluted at 4ºC overnight into 500 µL phosphate-
buffered saline (PBS).
• After being centrifuged for 4 minutes, the paper points/strips were
removed; both paper points/strips and supernatants were kept frozen
at -20̊C until assayed.
• Crevicular washes were obtained with following method
▫ A gel-loading capillary tip was carefully inserted into the crevices at a
level~1 mm below the gingival margin.
▫ In each case five sequential washes with 10µL of 0.9% NaCl were
performed using a micropipette.
▫ The washes were transferred into a microcentrifuged tube and centrifuged
for 4 minutes, after which supernatant were immediately frozen and kept
at-20̊C until analyzed.
• All samples containing blood were discarded and sampling was
repeated 2 days later.
10. MICROFLORA
• DNA was extracted using DNA-extraction system
according to recommendations of manufacturer from
paper points/strips after elution and 5µLof the washes.
• A real-time PCR was carried out using a real-time rotary
analyzer.
• PCR amplification was carried out in a reaction volume of 20
µL consisting of 2µL template DNA and 18 µL reaction mixture
composed of 2 µL PCR buffer , 2.75mM magnesium chloride,
0.2mM nucleotides.
• Negative and positive controls were included in each batch of
specimens.
• The positive control consisted of 2µL genomic DNA in
concentrations in range of 10^2 to 10^7 bacteria of the reference
strains, and negative control was 2µL of sterile water; each
control added 18µL reaction mixture.
• The cycling conditions comprised an initial denaturation step at
95 ̊C for 5 minutes followed by 45 cycles at 95 ̊C for 15 seconds
, at 65 ̊C for 20 seconds using a touhdown for five cycles and at
72 ̊C for 20 seconds.
11. NEUTROPHIL ELASTASE
• Neutrophil granulocyte elastase (NE) activity was
measured with a microplate assay using by using
chromogenic substrate N-methylsuccinyl-L-alanine-L-
proline-L-valine-para-nitroaniline.
• The assay was performed in a total volume of 150µL with a
0.75mM final substrate concentration in 50mM Tris-HCl, pH
7.5.
• The rate of p-nitroanilide released was recorded at 405 nm by
using microplate reader.
• One unit was calculated as the amount of enzyme that
hydrolyzes 1nmol substrate in 1 minute.
IL-6 and IL-8
• Concentration of IL-6 and IL-8 were determined by
commercially available enzyme-linked immunosorbent assay
(ELISA) kit as described in the manufacturer’s instruction.
• The detection level of kit was ~2pg/mL.
12. ANTIBODY USED IN ELISA ASSAY
• Antibodies (immunoglobulin Y [IgY]) anti-HRgpA
were raised in chickens as described by Pike et al.
• The IgY specific for the Rgp catalytic domain were
purified by affinity chromatography using
immobilized RgpB.
• Anti-RgpB mouse monoclonal antibody was produced
on site in a monoclonal facility.
• Because the caspase like domain of RgpB is essentially
identical with the catalytic domain of RgpA, the
obtained mAb reacted with equal affinity with both
Rgp.
13. LEVEL OF Rgp
• The level of the P.gingivalis proteases Rgp in the GCF was
determined by ELISA technique.
• 100µL from the paper GCF eluate and 10µL from each washing
GCF of diluted 10 fold to 100µL by addition of phosphate-
buffered saline was used.
• The wells of the microtiter plates were coated with chicken
antibody anti-Rgp in the final concentration of 1 µg/mL in
carbonate buffer(pH9.6) for 12 hrs at 4 ̊C.
• A negative control and GCF samples are added to wells and a
plate incubated for 12 hrs at 4 ̊C.
• After additional washing, secondary mAb anti-Rgp catalytic
domain was added to each well.
• The reaction was stopped by addition of 100µL of 1% H2SO4,
and absorbance was read at 450 nm.
14. IN VITRO STUDY
• In addition, in vitro experiment was performed to determine
the recovery levels of known concentrations of P.gingivalis,
AAC, IL-6 and IL-8, neutrophil elastase, and RgpB from paper
points.
• Bacteria was used in a range of 10^4 to 10^7 per microliter
• IL-6 and-8 were diluted to final concentrations of 62.5, 125, and
250pg/µL
• RgpB and human neutrophil elastase were tested at conc. 0.3 and 1.2
ng/µL and between 0.025 and 0.1µg/µL, respectively.
• The suspension or dilution media always contained 10% human
serum.
• 1µL of the final solution or suspension of tested bacteria or protein
was placed on point and paper strip.
• Further processing of the sample was made as described before for
clinical samples.
• An independent analysis was made at least in triplicate.
15. STASTICAL ANALYSIS
• Clinical data were expressed as mean standard
deviations.
• Laboratory variables were presented as median
including quartiles.
• Groups were compared using the paired Wilcxon test.
• The corelation among tested variables was made
using the Spearman test.
• Statistical software was used for all statistical
analyses.
16. RESULTS
• Subjects of all groups showed similar clinical signs; no difference
among groups was found to be statistically significant.
COMPARISON OF PAPER BASED METHODS
• The cytokines, IL-6 and IL-8 were detected more often when paper
strip were used compared to when paper points were used.
• The cytokines, IL-6 was measured in conc. <250pg/sites by mean
of paper strips and 154pg/site by mean of paper points (no
significance; P=0.311)
• IL-8 was analyzed insignificantly higher levels (P=0.001) by using
paper strips (<350pg/site) compared to paper points (246pg/site).
• The level of cytokines measured by both methods correlated
positively(IL-6:R=0.565, P<0.001; IL-8:R=0.337, P=0.0019).
• The neutrophil elastase activity was measurable in 85% of paper
strip sample and in 98%of paper point samples.
• Levels were slightly high when using paper points(not significant;
P=0.130)
17. • Measured bacterial load did not differ
significantly between the two sampling
method(.gingivalis: P=0.375; AAC:P=0.627)
• Two-third of the samples were positive for P.gingivalis
in contrast, AAC was detectable in 25% of paper point
samples and in 33%of the paper strip sample.
• Rgps were found in only two paper point eluates(4%)
and 11 paper strips eluates (22.9%)
• All Rgps positive samples were also positive for
P.gingivalis, which meant that Rgps was detected in
6%of positive samples by using paper points and in
35% by using paper strips.
18. Levels of IL-6 - and -8, activity of NE, bacterial loads of P. gingivalis and A.
actinomycetemcomitans, and levels of arginine-specific cysteine proteases (Rgps
derived from Pg) in GCF determined by using paper points and paper strips for
sampling materials. lg = logarithmic steps.
19. WASHING AS A SAMPLING METHOD
• Cytokines were detected in higher levels using the
washing method compared to the paper based methods.
These difference were more profound for IL-8(significant
difference compared to paper strips [P=0.012] and paper
points[P<0.001])
• The neutrophil elastase activity was significantly
lower in samples collected using the washing method
compared to paper strips (P=0.001)
• Bacterial organisms were found in low numbers, but
nevertheless, there was a good correlation to presence of
specific bacteria collected with other sampling
methods(p.gingivalis;P=0.004 compared to paper strips
and P-=0.001 compared to paper points; AAC:P=0.001
compared to paper strips ans P=0.002 compared to paper
point)
20. P.gingivalis AND ARGININE-SPECIFIC CYSTEINE
PROTEASE (Rgps)
• When comparing different methods of sampling, R-
gingipains were detectable in 49% of GCF washes, 13%of
paper-point samples, and 26% of paper-strips samples.
• To overcome limitations of individual methods, at least
partially, the higher values of each of the 144 sites
obtained by two methods were used for further analysis.
• Thus 70 sites (49%) tested positive for Rgp, and 109 sites
(76%) tested positive for P.gingivalis, which yielded a 64%
overlap of sites infected with P.gingivalis in which Rgps
were found.
• The analysis of all sites revealed that the concentration of
the Rgp gingipain correlated positively with bacterial load
of P.gingivalis (R=0.429; P<0.001)
21. Levels of arginine-specific cysteine protease (Rgps derived from P.gingivalis) in
GCF in relation to the load of P. gingivalis
22. IN VITRO EXPERIMENTS
• The mean recovery rate of cytokines was higher from
paper strips than from paper points.
• The difference was more significant for IL-8 compared to
IL-6.
• Concentration dependent defects were clearly visible.
• From paper strips, the lower concentration were
completely released; while in contrast, nearly 100% of Il-8
was eluted of 62.5 pg applied on paper strips, and at 250
pg applied on paper points, only 152 pg (60%) of cytokines
were recovered.
• Bacterial loads still attached to papers after elution were
found to be between 80.75% and 87.25%
• Bacterial load are normally counted as log states, and thus,
deviations appear to be smaller.
• Recovery rate of RgpB from paper was low, independent of
concentration of applied enzyme.
23. Recovered levels of cytokines applied in vitro onto paper points and paper strips.
24. DISCUSSION
• The quantity and quality of GCF are highly affected by method
• The wide range of volumetric distribution, the site-specific nature,
and the impact of a distinct sampling site on the volume were
described as important features of GCF collection and analysis.
• A standardization of the extent of probing depth, degree of gingival
inflammation, and distinct sampling may improve the reliability of
GCF methology.
• For this reason, only patients with comparable probing depths, good
oral hygiene (plaque index <0.35%),18 and low gingival
inflammation (samplecollection after the hygiene phase) to avoid
contamination of GCF samples with blood were chosen.
• A standardized time for collection of GCF by means of paper-based
methods was applied because the clinical situation was better
represented by the analysis of GCF based on the time of sampling
than on volume.
• This procedure allowed an instant freezing of samples to prevent
proteolysis.
• Nevertheless, the lack of sample standardization according to the
protein content or collected volume was a limitation of the study.
25. • Two paper-based sampling methods were compared using a regular
nitrocellulose paper point and a filter paper strip.
• These methods were quick and easy to use, were applied to individual sites,
and were not traumatic when correctly used.
• In addition, GCF was collected by an intracrevicular washing technique.
This technique uses the installation and continuous reaspiration of definite
solutions (e.g., Hanks’ balanced salt solution31 or PBS32) at the gingival
crevice.
• The method is highly sensitive, but requires participation of a trained,
experienced investigator to collect samples.
• The GCF collection with filter paper strips is probably the most preferred
sampling method.
• Several studies used this method to analyze the level of different cytokines
and other biomarkers in GCF. Significantly, amounts of IL-6 and -8 in GCF
reported in these studies were comparable with our data.
• Compared to the use of paper points, the use of filter paper strips resulted in
higher IL-8 levels. This finding was supported by the in vitro analysis; the
recovery rate of IL-8 was much lower from endodontic paper points
compared to paper strips.
• An earlier study also reported an incomplete recovery of proteins from
paper points supposedly because of binding of GCF proteins to the paper.
• The difference in elution of IL-6 and IL-8 was most likely due to difference
in the structure, charge distribution, and hydrophobicity of these cytokines
molecules.
26. • In addition, the recovery rate of the RgpB was only in the range of
23% to 26% of the predetermined concentrations.
• This low recovery of RgpB explained the low number of positive
samples collected by one of the paper-based methods.
• Intracrevicular washing was the only method that detected relevant
amounts of Rgps.
• Taking into account the molecular mass of Rgps and the volume of
GCF in periodontitis patients, a concentration <1.5 mM was found.
• This finding was highly significant because it allowed for the
prediction of whether a specific gingipains substrate would be
degraded in vivo.
• For example, a 150-fold less concentration would be sufficient to
cleave IL-6 and was more than high enough to destroy the
complement, protease inhibitors,8 and bactericidal peptides and
impair neutrophil functions in periodontal pockets.
• This underlined the importance of Rgps in vivo.
• Nevertheless, a correlation between the level of Rgps and the load of
P.gingivalis was not found.
• The levels of synthesized and released Rgps differ among strains and
depend on environmental conditions (e.g., the contact to epithelial
cells).
27. • In contrast to the superiority of the washing method in the
determination of gingipains, paper-based methods detected higher
levels of the NE activity.
• Earlier studies indicated that NE concentrations or activities in GCF
could be used to identify differences among disease activities within
patients.
• Therefore, this enzyme activity is an excellent qualitative measure of
gingival inflammation.
• Elastase is one of the proteolytic enzymes present in the
polymorphonuclear leukocyte (PMN) primary granules that is
released upon activation of the PMN and capable of degrading
extracellular matrix proteins of the connective tissue.
• The higher granulocyte elastase activity was previously observed in
patients with periodontitis (both aggressive and chronic) compared
to healthy controls.
• A positive correlation between the IL-8 level and NE activity was
found in the GCF of periodontitis patients, which was explained by
the intensity of the host inflammatory response induced by the IL-8–
elicited activity to activate granulocytes.
• Periodontal therapy reduced the levels of IL-8, suggesting a
relationship between this cytokine and periodontal status.
28. • The qualitative and quantitative composition of GCF with respect to
subgingival microbiota and host mediators is well known to reflect the
severity of periodontal disease.
• Unfortunately, the clinical significance of the analysis is often unclear
because different sampling methods are usually used to measure the
content of cytokines and to determine microflora of discrete periodontitis
sites.
• In the present study, the same sample was used for both analyses.
• Surprisingly, as shown in the in vitro assays, >80% of the bacteria were
still attached on the paper points or strips after the overnight elution with
PBS and before extraction of DNA.
• The pathogenic microflora was detected in nearly all patient samples with
both paper-based sampling methods.
• Papers are easy to insert into the gingival sulcus; the low costs of
endodontic paper points indicate their usage for determination
• of microflora only.
• The outcomes of paper points for microbiologic diagnostics were recently
compared to the sampling of subgingival biofilm with
• curets.
• The authors concluded that paper points were suitable for microbiologic
diagnostics.
29. • In this work, we found that supernatants of GCF washes were not
well suited for the determination of microflora.
• In the present study design, we added a centrifugation step to remove
cells and detect only soluble cytokines and NE in GCF so that bacteria
should not sediment, but they would if associated with host cells
and/or tissue debris.
• Indeed, the analysis of supernatants and sediments from five
additional GCF washing samples revealed that the majority of
bacteria were in pellets, and only <10% of bacteria were present in
supernatants.
• This result suggested that, before centrifugation, 5 mL of the washing
sample should be retained for microbiologic diagnostics.
• Keeping in mind the limitations of the method used for detection of
bacterial loads, it was not surprising that more samples were positive
for Rgps (total: 47 samples) than for P. gingivalis (total: 33 samples).
• The washing method was most suitable for detection of the Rgps.
• In comparison to paper-based sampling methods, the washing
method allowed for the collection of the highest amounts of
gingipains (significant difference: P = 0.018 for washes compared to
paper points).
30. CONCLUSIONS
• The washing technique is an alternative
sampling method of GCF for special purposes
when sampling by paper-based methods fails.
• Paper points are suitable for the determination
of the microflora and are recommended for daily
microbiologic analysis in dental practice.
• Paper strips are the method of choice for most
biomarkers in immunologic studies; a combined
determination of periodontopathic bacteria
seems possible.