Survival analysis of dental implants placed in a private practice. A multicenter prospective cohort study.

Background: This prospective cohort study aimed to assess the predictability and survival rates of dental implant treatment in edentulous patients while identifying potential factors contributing to implant failure. Material and Methods: A total of 80 outpatients, receiving 166 dental implants between September 2015 and November 2017 in two private dental clinics, were included in this study. Patient and implant characteristics, surgical procedures, primary stability, prosthetic rehabilitation, failure analysis, and survival rates were analyzed. Results: The majority of patients (53.75%) received a single implant for treating single-gap edentulism, with 6.25% requiring implants for fully edentulous jaws. Most implants (66.87%) were Avinent Ocean IC implants with specific design features. Surgical placement primarily occurred in healed pristine bone (78.31%), immediate implants in fresh extraction sockets (19.88%), and bone regeneration was simultaneous in 15.66% of cases. While 54.82% of implants achieved primary stability over 35Ncm, none exceeded 45Ncm, and only 4.82% failed to attain primary stability. Prosthetic rehabilitation revealed that 13.25% received immediate loading prostheses. During follow-up, four implants failed, resulting in a 2.41% failure rate, with bruxism (HR: 96.62; P< 0.001) and absence of primary stability (HR: 23.54; P< 0.001) significantly associated with implant failure. The cumulative survival rate at 24 months was 97.44%. Conclusions: This study demonstrates the high predictability and survival rates of dental implant treatment in edentulous patients, consistent with established standards. Factors such as bruxism and primary stability may impact early implant failure. Dental implants remain a reliable treatment option, boasting a 97.44% cumulative survival rate at 24 months. Further research is required to explore implant failure indicators and multifactorial influences. Key words:Dental implants, survival, edentulous patients.

Parents' Beliefs about Medicines and Their Influence on Inhaled Corticosteroid Adherence in Children with Asthma.

The most widespread chronic condition observed amid children globally is asthma. Only half of children with asthma adhere to their prescribed inhaled corticosteroids (ICS) therapy. Parents' emotions and perspectives regarding asthma have an impact on inhalation corticosteroid adherence. The participants in this study were 148 parents of children with asthma, with the aim to redintegrate their beliefs about medicines in general and specifically of ICS and the impact on ICS adherence in children with asthma. Children were mostly male (66.9%), older than five years (58.8%), parents were female, mean age 38, employed, and with a history of consumption of some form of corticosteroids. Parents' answers show that 50% of them disagreed with the statement that medicines are addictive, and 90% agree that medicine helps many to live better. A percentage of 77.7% of parents acknowledge that their child's health relies on inhaled corticosteroids (ICS), and 86.5% of parents agree that these medications safeguard their child from worsening health. Most of the parents (93.2%) adhere to the guidelines and instructions of the doctor. In summary, parents who hold the belief that medicines are neither overused nor harmful tend to exhibit a higher adherence. Furthermore, those with elevated adherence levels express lower levels of concern regarding the use of inhaled corticosteroids (ICS) in their children's asthma therapy.

Perception of Illness and Fear of Inhaled Corticosteroid Use among Parents of Children with Asthma.

The most prevalent children's chronic disease worldwide is asthma which has notable negative impacts on patients' and parent's quality of life. Daily inhaled corticosteroids (ICS) therapy is a preferred controller choice. This study was conducted on 148 parents of asthmatic children to establish parents' perception of illness and fear of inhaled corticosteroids using B-IPQ and TOPICOP questionnaires. Children were in the majority male (66.9%), older than five years (58.8%), with comorbidities, and family history of atopy. Parents were female, with a mean age of 38, employed, and with a history of some form of corticosteroid use. Most parents were not afraid of ICS usage (71.6%). Unemployed parents and parents who had no medical education had a statistically significantly higher fear of using ICS (p = 0.002, p = 0.03). A child's illness affects the parents' lives and parents who are afraid of using ICS react more emotionally to the child's illness. Better understanding and less concerned about child's disease are parents of children with controlled asthma. The parents' perspective of children's asthma will affect the duration and dose of ICS treatment they will give to their children and directly influence the level of asthma control.

CHARACTERISTICS AND TREATMENT OUTCOMES OF UROLOGIC PATIENTS ADMITTED TO THE INTENSIVE CARE UNIT, OSIJEK UNIVERSITY HOSPITAL CENTER.

The postoperative care unit at the Department of Urology has significantly improved treatment of patients undergoing surgical procedures and reduced admission of urologic patients to the Intensive Care Unit (ICU). We examined the characteristics of urologic patients, time on mechanical ventilation, most common complications, and mortality in the period from January 2017 to March 2022. A total of 84 admissions to ICU were recorded, accounting for 1.5% of all patients having undergone surgical, therapeutic or diagnostic interventions under general or regional anesthesia at the Department of Urology. The most common reasons for admission to ICU were respiratory failure (79 patients), hemodynamic instability, and bleeding. The median time on mechanical ventilation was 9.7 [2.4-58.2] hours in urology patients vs. 6 [3-14.7] hours in the rest of surgical ICU patients (p=0.058). Hypertension and renal failure were more common in urologic than in the rest of surgical ICU patients (p<0.05). The overall mortality of urologic patients was lower than in the rest of surgical ICU patients (10.7% vs. 18.99%, p=0.08) but the difference did not reach statistical significance. Independently of the lower mortality, improvements in the outcome of urologic patients admitted to the ICU are feasible. Early identification of patients at risk of infections, postoperative respiratory failure, cardiovascular incidents, and bleeding may further reduce mortality and improve outcomes.

Acute Arterial Thrombosis of Lower Extremities in COVID-19 Patients.

Clinical signs and symptoms of COVID-19 varied from asymptomatic forms to severe, life-threatening conditions that required treatment in intensive care units. These severe forms of illness are connected with a hypercoagulable state due to excessive inflammation, hypoxia, immobilisation, and altered angiotensin-converting enzyme 2 (ACE-2). In total, 17 COVID-19 positive patients were diagnosed with peripheral arterial thrombosis (AT), 13 of them had COVID-19 pneumonia. Laboratory findings in patients with X-ray confirmed pneumonia showed a four times higher neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP) and three times higher lactate dehydrogenase level (LDH) than patients without confirmed pneumonia. Patients with pneumonia had significantly more bilateral occlusions of the lower extremities and a significantly higher percentage with complete occlusion of the arteries than patients without pneumonia. The rate of limb loss was 35.3%. They were all from the group with COVID-19 pneumonia. Ten out of thirteen patients with pneumonia died due to acute respiratory distress syndrome (ARDS). All patients without pneumonia were discharged from the hospital. The aim of this retrospective study was to report the incidence of arterial thrombosis of lower extremities and their complications in the acute phase of the infection among COVID-19 patients admitted to the hospital for treatment.

Knowledge of Dental Students from Croatia, Slovenia, and Bosnia and Herzegovina about Dental Care of Oncology Patients.

The central role of the dentist in the treatment of oncology patients is to care for the patient's oral cavity before, during, and after radio/chemotherapy. The aim of this research was to determine the knowledge of dental students from five universities in three neighboring countries, Croatia (Split, Rijeka, and Zagreb), Bosnia and Herzegovina (Sarajevo), and Slovenia (Ljubljana), about oncology patients' dental care. A total of 140 students in their fourth, fifth, and sixth year of dental medicine studies participated in this research. A questionnaire with 36 specific questions was designed for this research and included questions about dental care of oncologic patients before, during, and after the oncology therapy. Most students are familiar with the incidence and most common type of head and neck tumors, while knowledge about tumor treatment and the side-effects of radiation therapy and/or chemotherapy is weak. Students did not show satisfactory knowledge about osteoradionecrosis, which is the most serious side-effect of radiotherapy; therefore, the emphasis on additional education should be greatest in this area. Teaching staff should be aware of lack of student knowledge and try to offer more information and practice in providing dental care for oncology patients.

A measurement of irradiance of light-curing units in dental offices in three Croatian cities.

Aim To determine irradiance of light-curing units (LCUs) in dental offices in three Croatian cities and to compare irradiance values with the age and model of LCUs. Methods Private and public dental offices in three most prominent cities in Croatia (Rijeka, Split and Zagreb) were included in this study. In total, 195 LCUs were tested, using radiometer Ivoclar Bluephase Meter 2 for irradiance (mW/cm2 ). The minimum acceptable value was set at 400 mW/cm2 . The age, model and difference between declared and measured irradiance of the LCUs were also determined. Of the total of 195 LCUs, 190 (98%) were LED (light-emitting diode) and 5 (2%) were QTH (quartz-tungsten - halogen). Results The mean age of tested LCUs was 4.43±3.4 years; the oldest was in Rijeka, 5.2±3.8 years. The overall mean irradiance for all three cities was 806.4 mW/cm2 (p=0.0004). Of all LCUs, 11.3% were considered clinically unacceptable with irradiance of less than 400 mW/cm2 . Of all tested LCUs 42% (p=0.0005) had a 30% lower value of irradiance than the manufacturer of the LCU declared. In 73% tested LCUs, there was a matching between measured and declared irradiance. The age and model of LCUs had the most significant impact on irradiance. Conclusion The most commonly used LCU included in dental offices was LED. Mean irradiance was good enough to secure adequate polymerization of resin-based materials. Irradiance decreases with usage time of LCU.

Spatio-Temporal Expression Pattern of Ki-67, pRB, MMP-9 and Bax in Human Secondary Palate Development.

We analyzed the immunohistochemical expression of Ki-67, pRb, Bax, and MMP-9 during the human secondary palate formation (7th to 12th developmental weeks (DWs). The most significant proliferation was observed in the seventh DW with 32% of Ki-67-positive cells in the epithelium, while loose ectomesenchyme condensations (lec) and loose non-condensing ectomesenchyme (lnc) had only 18 and 11%, respectively (Kruskal-Wallis, p < 0.001), and diminished afterwards. Contrarily, pRb-positive cells were mostly located in the lnc (67%), with significant difference in comparison to epithelium and lec in all investigated periods (Kruskal-Wallis, p < 0.001). Ki-67- and pRb-positive cells co-expressed occasionally in all investigated periods. MMP-9 displayed a strong expression pattern with the highest number of positive cells during the seventh DW in the epithelium, with significant difference in comparison to lec and lnc (Kruskal-Wallis, p < 0.0001). The ninth DW is particularly important for the Bax expression, especially in the epithelium (84%), in comparison to lec (58%) and lnc (47%) (Kruskal-Wallis, p < 0.001). The co-expression of Bax and MMP-9 was seen only in the epithelium during seventh and ninth DWs. Our study indicates the parallel persistence of proliferation (Ki-67, pRb) and remodeling (MMP-9) that enables growth and apoptotic activity (Bax) that enable the removal of the epithelial cells at the fusion point during secondary palate formation.

Students' Practices and Knowledge on Antimicrobial Usage and Resistance in Split, Croatia: The Education of Future Prescribers.

Aims: To evaluate the effect of biomedical students' ongoing education, we assessed their knowledge and attitudes toward antimicrobial use. Study Design: A cross-sectional study was carried out among the students of four study programs: Medicine in Croatian, Medicine in English, Dental medicine, and Pharmacy. The anonymous questionnaire was distributed to students who attended classes from April to May 2018. Results: A total of 947 (86%) out of 1,107 students enrolled at the University of Split School of Medicine participated in this study. A third of dental students (51/159) and a quarter of medical (113/458) and pharmacy students (32/130) believed that paracetamol was an antibiotic that reduces pain. However, the percentage significantly decreased from the first to the final years. Only 31% of the final year dental medicine students (5/16) named a correct guideline for the usage of antimicrobial drugs, 23% of medical students (18/78), and none in the English program. Pharmacy students were the most informed, since 76% of the final year students (16/21) named Intersectoral Coordination Mechanism for the Control of Antimicrobial Resistance (ISKRA) guidelines. Conclusion: The students showed poor knowledge on the use of guidelines for antibiotic use, highlighting the need for changes in the existing curricula, including a more effective course on antimicrobial prescribing.

Antimicrobial effectiveness of polyhexamethylene biguanide on Enterococcus faecalis, Staphylococcus epidermidis and Candida albicans.

Aim To evaluate the antimicrobial activity of 0.2% polyhexamethilene biguanide (PHMB) in root canal models infected with Enterococcus faecalis, Candida albicans and Staphylococcus epidermidis. PHMB was compared in these tests with 2.5% NaOCl and 0.2% CHX. Methods Prepared models of 50 human root canals (n=50) were immerged in mixed, four- weeks old culture that consisted of E .faecalis, S. epidermidis and C. albicans. Roots were randomly divided into three groups: one with 30 (n=30) and two with 10 (n=10) samples. Samples were treated with polyhexamethylene biguanide (PHMB) (0.2%), sodium hypochlorite (NaOCl) (2.5%) and chlorhexidine (CHX) (0.2%), respectively. Root dentin was sampled before and after the tretment with these solutions. Colony- forming units (CFU) were counted to asses the antimicrobial effects of three solutions on viability of selected microrganisms in specimens before and after the treatment.T-test was used for comparison of results between specimens before and after the treatment, while Newman-Keuls test was used for pairwise comparison at p=0.05. Results The PHMB was significantly more efficient in reducing the number of all three tested microorganisms. NaOCl and CHX made only statistically significant (p<0.05) difference in case of E. faecalis and S. epidermidis. In the case of C. albicans, this difference was not statistically significant due to the small number of positive samples and high initial dispersion of results. Conclusion Both solutions PHMB and NaOCl were successful in eliminating E. faecalis and S. epidermidis from the mature dentin biofilm, CHX was not successful enough.

Self Assessment of Dental students' Perception of Learning Environment in Croatia, India and Nepal.

AIM: The aim of this study was to assess dental students' perception of different learning environment in India, Nepal and Croatia. METHODS: The study was conducted during a period of academic year 2016/17. A total of 849 dental students participated in the study. There were 188 respondents from Croatia, 373 from India, and 288 from Nepal. Non-responders were not followed up. The Dental Student Learning Environment Survey (DSLES) was used which consisted of 55 items subdivided into seven scales. The scales measured the following perceptions: Flexibility, 'Student-to-Student Interactions, Emotional Climate, Supportiveness, Meaningful Experience, Organization and Breadth of Interest. Statistical analysis of the data utilised the Kolmogorov Smirnov test. The Kruskal-Wallis "non-parametric ANOVA" was also used to test the differences between the countries. A post hoc analysis was performed using Ranks tables and the Median test. RESULTS: The response rate was 26.9%. Significant differences between the countries were found for all DSLES variables (Kruskal-Wallis, p<0.01). The Median test also showed significant differences between the countries for all DSLES variables (p<0.01). The scales with the highest mean values were 'Student-to-student interactions' in India and Nepal, and the 'Emotional Climate' in Croatia. CONCLUSIONS: Students in Croatia rated their school only with grades excellent and good, while their colleagues in India and Nepal were more critical. Despite the different settings in three countries, 'Flexibility' was identified as the area of weakness in all three educational systems.

Involvement of IGF-2, IGF-1R, IGF-2R and PTEN in development of human tooth germ - an immunohistochemical study.

Insulin-Like Growth Factor 2 (IGF-2) is a peptide hormone essential for prenatal growth and development. IGF-2 exerts its mitogenic effects via Insulin-Like Growth Factor 1 Receptor (IGF-1R), and is eliminated by binding to Insulin-Like Growth Receptor 2 (IGF-2R). IGF-2 is also negatively regulated by Phosphatase and Tensin Homolog (PTEN), a phosphatase mutated in various tumors. Not much is known about the interplay between these factors during human odontogenesis. In this study, expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN were analyzed by double immunofluorescence in incisor human tooth germs during the foetal period of development between the 7th and 20th gestational week. Throughout the investigated period, IGF-2 was mostly expressed in enamel organ, whereas mild to moderate expression of PTEN could be seen in dental papilla and parts of enamel organ. Expression of IGF-1R was ubiquitous and displayed strong intensity throughout the entire enamel organ. In contrast, expression of IGF-2R had rather erratic pattern in enamel organ and dental papilla alike. Expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN in highly proliferative cervical loops, as well as in differentiating pre-ameloblasts and pre-odontoblasts of cusp tip region during the early and late bell stages when enamel organ acquires definitive shape, indicate importance of these factors in crown morphogenesis of human incisor. Taken together, our data suggest the involvement of IGF-2, IGF-1R, IGF-2R and PTEN in temporo-spatial patterning of basic cellular processes (proliferation, differentiation) during normal tooth development. They are also relevant for improving knowledge of molecular basis of human odontogenesis.

The prevalence of celiac disease in patients with geographic tongue.

BACKGROUND: Geographic tongue (GT) is a benign inflammatory condition usually involving the dorsal surface and lateral borders of the tongue. Numerous etiological factors of GT have been suggested, including immunological factors; genetic; atopic or allergic predisposition; emotional stress; and hormonal disturbances. GT may also coexist as one of the possible manifestations of celiac disease (CD). Therefore, the aim of this study was to investigate the prevalence of CD, positive serologic tests for CD screening, and HLA-DQ presence in patients with GT. METHODS: Tissue transglutaminase antibodies (anti-tTG), antibodies against gliadin (AGA), and human leukocyte antigen (HLA) typing were assessed for 60 GT patients and 60 healthy control subjects. The duodenal biopsy was performed in patients with positive serologic tests. RESULTS: We found that 9 (15%) GT patients were positive for IgA tTG, and in those patients histological changes consistent with CD were confirmed by duodenal biopsy. Only two of them reported the presence of gastrointestinal symptoms. There were statistically significant differences between the GT patients and control group for immunoglobulin (Ig) A tTG (P = 0.03), IgG tTG (P = 0.04), IgA AGA (P = 0.04), and IgG AGA (P = 0.02). CONCLUSION: The results of our study demonstrated the increased prevalence of CD in patients with GT. Therefore, the clinical oral examination should be considered a diagnostic tool, especially in atypical or silent forms of CD, since it may contribute to provide an early diagnosis.

Analysis of expression patterns of IGF-1, caspase-3 and HSP-70 in developing human tooth germs.

AIMS: To analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis. MATERIALS AND METHODS: Head areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker's expression pattern was also performed. RESULTS: During the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period. CONCLUSIONS: Expression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops.

Oral health changes during early phase of orthodontic treatment.

The aim of the study was to assess the influence of fixed orthodontic appliance on Streptococcus (S.) mutans and S. sobrinus counts in orthodontic patients with regard to their previous caries experience (Decayed, Missing and Filled Teeth (DMFT) index) during the first 12 weeks of orthodontic treatment. Twenty-two patients that satisfied inclusion criteria (healthy systemic and periodontal condition, avoidance of antibiotic therapy and antiseptic mouthwashes in the past three months) were included. All clinical measurements took place prior to and 12 weeks after fixed orthodontic appliance placement, in the following order: 1) stimulated saliva flow (SS); 2) Simplified Oral Hygiene Index (OHI-S); and 3) DMFT. The method of polymerase chain reaction (PCR) was used to detect the presence of S. mutans and S. sobrinus at T1 and T2. T-test showed significant increase in DMFT index and SS between T1 and T2. Results also indicated significant improvement in OHI-S index. By use of the PCR method, S. mutans was detected in two patients at T1. At T2, two more patients had S. mutans, but the increase was not statistically significant. Using the same method, S. sobrinus was detected only in two patients at T2. In conclusion, fixed orthodontic appliances did not induce statistically significant changes in caries microflora even in the presence of enhanced oral hygiene habits.

Prevalence of third molars and pathological changes related to them in dental medicine.

The purpose of this study was to determine the prevalence of third molars in terms of respondents' age. Furthermore it was desired to determine the extent of the manifestation of pathological changes, such as caries, apical lesions, and alveolar bone resorption. This study was based on 500 OPGs of patients from the Faculty of Dentistry, University of Zagreb. Patients were divided into seven age groups. The total number of molars, the number of cavities on remaining molars, the quality of endodontic treatment and the evaluation of their performance and presence of periapical lesions was analyzed. As apart of the periodontal status, the number of inadequate fillings, the number of damaged furcations and bone resorption was analyzed. The statistical analysis was performed by means of STATISTICA 7 statistical package (StatSoft, Tulsa, USA). The correlation between the total number of molars and the age of patients was analyzed by means of Spearman's correlation to the level of significance of 0.05. The number of first, second and third molars significantly decreased in higher age groups. The prevalence of caries in third molars as compared to first and second molars was generally lower, third molars were endodontic treated at least. Assessment of involved furcations showed that the lowest percentage of 8% was found in third molars. The majority of all remaining molars had bone resorption of 1-3 mm.

Genotoxic biomonitoring of flowable and non-flowable composite resins in peripheral blood leukocytes.

OBJECTIVE: Composite restorative materials represent one of the most important groups of materials in contemporary dental practice. However, their incomplete polymerization may lead to monomer-induced genotoxicity. The objective of this study was to evaluate the genotoxicity of three flowable (Filtek Supreme XT Flow, Tetric EvoFlow, Gradia Direct Flo) and three non-flowable dental composite materials (Filtek Z250, Tetric EvoCeram, Gradia Direct Posterior). MATERIALS AND METHODS: Genotoxicity assessment of composite materials was carried out in vitro in human peripheral blood leukocytes using the alkaline single-cell gel electrophoresis technique (comet assay). Prepared materials were eluted in saline solution for 1 h, 1 day and 5 days. Thereafter leukocyte cultures were treated with different concentrations of eluates obtained from each of the tested dental composite materials. Kruskall-Wallis non-parametric test was used for statistical analysis (p < 0.05). RESULTS: The tested materials did not show genotoxic effects after exposure of leucocytes to 1 h eluates. Culture treated with 1 day eluates of all tested materials, only at a highest concentration (10(-2)), affected the measured cytogenetic parameters. Of all tested materials, only Filtek Z250 and Filtek Supreme XT Flow did not exhibit a genotoxic effect in cultures that were under the influence of 5 day eluates. CONCLUSION: Tested materials exhibited limited genotoxic activity in peripheral blood leukocytes. Since the effect was observed only in leukocyte cultures treated by 1-day eluates at the highest concentration (10(-2)) and it decreases in cultures exposed to 5 day eluates, it should not pose a significant risk to the human genome.

Whole organism high-content screening by label-free, image-based Bayesian classification for parasitic diseases.

Sole reliance on one drug, Praziquantel, for treatment and control of schistosomiasis raises concerns about development of widespread resistance, prompting renewed interest in the discovery of new anthelmintics. To discover new leads we designed an automated label-free, high content-based, high throughput screen (HTS) to assess drug-induced effects on in vitro cultured larvae (schistosomula) using bright-field imaging. Automatic image analysis and Bayesian prediction models define morphological damage, hit/non-hit prediction and larval phenotype characterization. Motility was also assessed from time-lapse images. In screening a 10,041 compound library the HTS correctly detected 99.8% of the hits scored visually. A proportion of these larval hits were also active in an adult worm ex-vivo screen and are the subject of ongoing studies. The method allows, for the first time, screening of large compound collections against schistosomes and the methods are adaptable to other whole organism and cell-based screening by morphology and motility phenotyping.

Quantification of lipid droplets and associated proteins in cellular models of obesity via high-content/high-throughput microscopy and automated image analysis.

Intracellular lipid droplets are associated with a myriad of afflictions including obesity, fatty liver disease, coronary artery disease, and infectious diseases (eg, HCV and tuberculosis). To develop high-content analysis (HCA) techniques to analyze lipid droplets and associated proteins, primary human preadipocytes were plated in 96-well dishes in the presence of rosiglitazone (rosi), a PPAR-(c) agonist that promotes adipogenesis. The cells were then labeled for nuclei, lipid droplets, and proteins such as perilipin, protein kinase C (PKC), and hormone-sensitive lipase (HSL). The cells were imaged via automated digital microscopy and algorithms were developed to quantify lipid droplet (Lipid Droplet algorithm) and protein expression and colocalization (Colocalization algorithm). The algorithms, which were incorporated into Vala Science Inc's CyteSeer((R)) image cytometry program, quantified the rosi-induced increases in lipid droplet number, size, and intensity, and the expression of perilipin with exceptional consistency (Z' values of 0.54-0.71). Regarding colocalization with lipid droplets, Pearson's correlation coefficients of 0.38 (highly colocalized), 0.16 (moderate), and -0.0010 (random) were found for perilipin, PKC, and HSL, respectively. For hepatocytes (AML12, HuH-7, and primary cells), the algorithms also quantified the stimulatory and inhibitory effect of oleic acid and triacsin C on lipid droplets (Z's > 0.50) and ADFP expression/colocalization. Oleic acid-induced lipid droplets in HeLa cells and macrophages (THP-1) were also well quantified. The results suggest that HCA techniques can be utilized to quantify lipid droplets and associated proteins in many cell models relevant to a variety of diseases.

Plasma membrane assays and three-compartment image cytometry for high content screening.

High throughput image cytometers analyze individual cells in digital photomicrographs by first assigning pixels within each image to plasma membrane, cytoplasm, nucleus, or other regions. In this study, we report on a novel algorithm that: 1) identifies plasma membrane regions to measure changes in plasma membrane-associated proteins (protein kinase C [PKC] alpha, N-cadherin, E-cadherin, vascular endothelium [VE]-cadherin, and pan-cadherin) that regulate cell division, migration, and adhesion and 2) delineates the cell for generalized three-compartment image cytometry. Validation assays were performed for these proteins on cells cultured in 96-well plates and also for tissue sections obtained from transgenic and chemical carcinogenic models of skin cancer. The algorithm successfully quantified phorbol 12-myristate 13-acetate (PMA)-induced plasma membrane localization of PKCalpha in HeLa cells (Z' of 0.88). Additionally, PMA activated translocation to the plasma membrane at P < .01 of N-cadherin (in HeLa cells), E-cadherin (in A431 cells), and VE-cadherin (in human dermal microvascular endothelial cells), suggesting a relationship between PKCalpha activity and cadherin localization. For VE-cadherin, a Z' of 0.52 was obtained between serum-free medium, which increased VE-cadherin, and EGTA, which diminished VE-cadherin at the plasma membrane. For sections obtained from the transgenic skin cancer model, analysis of images with the plasma membrane algorithm revealed that tumor cells exhibited cadherin expression that was just 34% of that expressed by surrounding normal tissue; furthermore, tumor cells expressed elevated DNA content, consistent with development of aneuploidy. In contrast, increased DNA content did not occur for tumor cells produced by chemical carcinogenesis. The results demonstrate that this new algorithm for plasma membrane image cytometry enables statistically significant analyses in a variety of applications in both cultured cells and tissue sections.