Increased expression of ubiquitin C-terminal hydrolase L1 in astrocytomas of ascending grades.

BACKGROUND: The ubiquitin-proteasome pathway controls the monitoring and degradation of important proteins and is involved in several cellular processes, such as development, differentiation, and transcriptional regulation. Recent evidence has shown that ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), a member of the deubiquitinating enzyme family that removes ubiquitin from protein substrates, is overexpressed in many types of cancer. AIM: This study thus examined the expression of UCH-L1 in human astrocytoma tissues. MATERIAL AND METHODS: Formalin-fixed, paraffin-embedded astrocytoma samples were obtained from 40 patients, after which histopathological examination, typing, and grading were performed. The study group included 10 histologically normal brain tissues, which served as the control group, and 10 WHO grade II, 10 WHO grade III, and 10 WHO grade IV (glioblastoma) samples. Normal brain tissue samples were obtained from the histologically normal, non-tumoral portion of the pathology specimens. UCH-L1 expression was evaluated using quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: Astrocytoma tissues exhibited higher UCH-L1 expression compared to the control group. UCH-L1 overexpression increased significantly together with the increase in astrocytoma grades (from II to IV). CONCLUSION: UCH-L1 could be a good diagnostic and therapeutic marker for determining astrocytoma development and progression.

How do endoscopic bladder tumor resection techniques affect pathology practice? EAU Section of Uro-Technology (ESUT) and Uropathology (ESUP) survey.

PURPOSE: We aimed to examine how different endoscopic bladder tumor resection techniques affect pathologists' clinical practice patterns. METHODS: An online survey including 28 questions clustered in four main sections was prepared by the ESUT ERBT Working Group and released to the pathologists working in the institutions of experts of the ESUT Board and the working groups and experts in the uropathology working group. A descriptive analysis was performed using the collected data. RESULTS: Sixty-eight pathologists from 23 countries responded to the survey. 37.3% of the participants stated that they always report the T1 sub-staging. Of those who gave sub-staging, 61.3% used T1a, b. 85.2% think that en bloc samples provide spatial orientation faster than piecemeal samples, and 60% think en bloc samples are timesaving during an inspection. 55.7% stated that whether the tissue sample is en bloc or piecemeal is essential. 57.4% think en bloc sample reduces turnaround time and is cost-effective for 44.1%. A large number of pathologists find that the pathology examination of piecemeal samples has a longer learning curve. CONCLUSION: The survey shows that pathologists think that they can diagnose faster, accurately, and cost-effectively with ERBT samples, but they do not often encounter them in practice. Moreover, en bloc samples may be a better choice in pathology resident training. Evidence from real-life observational pathology practice and clinical research can reveal the current situation more clearly and increase awareness on proper treatment in endoscopic management of bladder tumors.

Histopathological Evaluation of Platelet-Rich Plasma Effect in Acute Tympanic Membrane Perforation.

Platelet-rich plasma (PRP) is a reliable and has low side-effect profile and has beneficial effects on wound healing. Its investigatory effects on wound-healing process were shown on various tissues. This study aims to investigate PRP's local application effects to perforated rat TM in terms of healing and histopatological outcomes. Twenty-two Wistar rats were used in the study. The rats' ears were examined with a pediatric endoscope (2.7 mm, 0°), and the TM posterior quadrant of their right ear was perforated with a 20-gauge needle. After this procedure, the rats were divided into two equal groups. A spongel with PRP was applied on the perforated TM in the first group, and spongel with standard saline solution was applied on the second group. Following the sacrifice, the middle air bullas were carefully dissected and removed for histopathological examination. Hematoxylin eosin (for fibroblasts, lymphocyte, collagen fibers) and immunohistochemical staining were done for epithelial growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR1), and vascular endothelial growth factor (VEGF) staining for histopathologic examinations. There was not a significant difference between the two groups for lymphocyte. There was a significant difference between control and study groups for collagen and EGFR (P < 0.05). Although the mean value of FGF- and VEGF-positive cells was higher in the study group than in the control group, the difference was not significant (P > 0.05). PRP is an effective autologous material for the healing process of acute TM perforations in a rat model, as demonstrated in the present study. We think that the use of PRP for acute TM perforations can have a positive effect on the healing process by increasing the level of growth factors, especially EGFR. In addition, an increase in collagen can also have a positive effect on healing. Supplementary Information: The online version contains supplementary material available at 10.1007/s12070-021-02912-2.

Myxoma Arising from the Middle Ear: A Case Report.

The primary head and neck myxomas are rare, generally arising from the mandible, maxilla, and oral cavity. Other anatomical areas, such as cardiac myxomas, may also have metastases to the head and neck regions. The middle ear is an extremely rare location for myxomas. Myxoma slowly grows and is usually asymptomatic until it affects the surrounding structures. Surgical treatment is performed with a complete en bloc resection where possible. We report a case of a 42-year-old woman with myxoma arising from the right middle ear because of her tumor's rare anatomical region. Her main complaints were progressive fullness and loss of hearing which she felt for approximately 1 year on the right ear. High-resolution computed tomography (HRCT) revealed an isodense soft tissue mass localized in the right mastoid bone and the middle ear. The mass was totally removed by canal wall up tympanomastoidectomy. At the last follow-up examination on 36 months after the surgery, the patient was asymptomatic, and there were no signs of recurrence.

The Use of Radiofrequency Thermal Ablation Method in The Treatment of Hepatic Hydatid Cysts: Ex vivo Sheep Study

Objective: Hydatid disease is a disease caused by parasites belonging to the echinococcus family. This disease is often caused by Echinococcus granulosus and rarely by echinococcus alveolaris.The parasite may cause illness anywhere in the human body, mainly in liver. In this study, we aimed to destroy the hydatid cyst viability by Radiofrequency Thermal Ablation (RFTA) method which has been used in many areas in medicine. Methods: We used fresh sheep liver with hydatid cysts. Average diameter of cysts was 3.3 cm. The study was performed in 3 groups, each of which involved 20 cysts. After more than half of the cyst fluid was drained, ablation was performed. When the core temperature of the cyst exceeded 95°C, ablation procedure was continued for 3 minute in 1st group and for 4 minutes in 2nd group. Third group was the control group. And then, cyst fluid and germinative membrane were collected for microbiologic and pathologic assessment. Results: In 1st group, the cysts could not be destroyed at the desired level. In 2nd group, it was observed that 100% of the protoscolex died and 100% of the germinative membranes was degenerated. In control group, %13 of protoscolex died and %10 of germinative membranes wasdegenerated. Conclusion: We destroyed all the protoscolex and germinative membranes by using RFTA in 2nd group.

New Clues in the Malignant Progression of Glioblastoma: Can the Thioredoxin System Play a Role?

AIM: To evaluate and compare the expression of thioredoxin reductase 1 (TrxR1) in primary and secondary glioblastoma samples. MATERIAL AND METHODS: Surgically resected human glioblastoma samples from 40 patients who underwent surgery at our institution were extracted from their histopathological specimens and divided into three groups. Ten histopathologically regular cerebral tissue samples, acquired from the non-neoplastic portion of the specimens, were assigned as the control group. Twenty specimens that included tumoral tissue from each type of glioblastoma (WHO grade IV, primary and secondary) were assigned as the primary and secondary glioblastoma groups. TrxR1 expression was analyzed by using both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Isocitrate dehydrogenase 1 (IDH1) mutation was analyzed by immunohistochemistry. Ki-67 proliferative index and apoptosis were also analyzed by immunohistochemistry. The differences between the groups were statistically compared and the correlation between these parameters was analyzed. RESULTS: The expressions of TrxR1 and Ki-67 values were significantly higher in primary glioblastoma. IDH1 mutation was significantly higher in secondary glioblastoma. TrxR1 expression was found to be highly correlated with the Ki-67 index. The apoptotic index was similar between primary and secondary glioblastoma. CONCLUSION: This study showed a high TrxR1 expression in primary glioblastoma which could indicate a role of the Trx system in promoting the malignant progression by some complex processes.

The Prevalence of Fabry Disease in Patients with Chronic Kidney Disease in Turkey: The TURKFAB Study.

BACKGROUND/AIMS: Fabry disease is a treatable cause of chronic kidney disease (CKD) characterized by a genetic deficiency of α-galactosidase A. European Renal Best Practice (ERBP) recommends screening for Fabry disease in CKD patients. However, this is based on expert opinion and there are no reports of the prevalence of Fabry disease in stage 1-5 CKD. Hence, we investigated the prevalence of Fabry disease in CKD patients not receiving renal replacement therapy. METHODS: This prospective study assessed α-galactosidase activity in dried blood spots in 313 stage 1-5 CKD patients, 167 males, between ages of 18-70 years whose etiology of CKD was unknown and were not receiving renal replacement therapy. The diagnosis was confirmed by GLA gene mutation analysis. RESULTS: Three (all males) of 313 CKD patients (0.95%) were diagnosed of Fabry disease, for a prevalence in males of 1.80%. Family screening identified 8 aditional Fabry patients with CKD. Of a total of 11 Fabry patients, 7 were male and started enzyme replacement therapy and 4 were female. The most frequent manifestations in male patients were fatigue (100%), tinnitus, vertigo, acroparesthesia, hypohidrosis, cornea verticillata and angiokeratoma (all 85%), heat intolerance (71%), and abdominal pain (57%). The most frequent manifestations in female patients were fatigue and cornea verticillata (50%), and tinnitus, vertigo and angiokeratoma (25%). Three patients had severe episodic abdominal pain attacks and proteinuria, and were misdiagnosed as familial Mediterranean fever. CONCLUSIONS: The prevalence of Fabry disease in selected CKD patients is in the range found among renal replacement therapy patients, but the disease is diagnosed at an earlier, treatable stage. These data support the ERBP recommendation to screen for Fabry disease in patients with CKD of unknown origin.

Intratumoral hemorrhage-related differences in the expression of vascular endothelial growth factor, basic fibroblast growth factor and thioredoxin reductase 1 in human glioblastoma.

The present study was designed to evaluate the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and thioredoxin reductase 1 (TrxR1) in glioblastoma multiforme (GBM) with and without intratumoral hemorrhage. Surgically resected human GBM samples from 20 patients who underwent surgery at our institute were extracted from the histopathological specimens and divided into two groups. A total of 10 samples from each type of GBM (World Health Organization grade IV, intratumoral hemorrhage-positive or -negative) were included in each group. VEGF, bFGF and TrxR1 expression was analyzed using immunohistochemistry and the results were compared between groups. VEGF and bFGF immunoreactivity was significantly higher in GBMs containing intratumoral hemorrhage. Furthermore, VEGF, bFGF and TrxR1 immunointensity was significantly higher in GBMs containing intratumoral hemorrhage. Thus, the present study demonstrated a higher VEGF, bFGF and TrxR1 expression in GBMs contain intratumoral hemorrhage, indicatiogn a role of VEGF, bFGF and TrxR1 expression in the promotion of tumoral angiogenesis and tumoral growth by complex mechanisms that require further elucidation.

Telmisartan ameliorates oxidative stress and subarachnoid haemorrhage-induced cerebral vasospasm.

OBJECTIVES: Growing evidence suggests that oxidative stress is one of the factors contributing to subarachnoid haemorrhage (SAH)-induced cerebral vasospasm. SAH-induced cerebral vasospam alters thioredoxin (Trx) cycle enzymes and thioredoxin-interacting protein (TXNIP) as an important endogenous antioxidant system. In this study, we have explored the effects of telmisartan on the vascular morphological changes, endothelial apoptosis, tissue oxidative stress status and the level of Trx cycle enzymes/ TXNIP in a rabbit SAH model. METHODS: Forty male New Zealand rabbits were randomly divided into five groups of eight rabbits each: control group, sham group, SAH group, SAH + vehicle group and SAH + telmisartan group. SAH was created by a single cisterna magna blood injection. SAH + telmisartan group received telmisartan treatment (5 mg/kg intraperitoneal, once daily) for 72 h. The brainstem tissue Trx1, Trx2, Trx reductase (TrxR), TrxR1and TXNIP levels were investigated. Total oxidant status (TOS), total antioxidant status (TAS), malondialdehyde (MDA) levels and tumour necrosis factor alpha (TNF alpha) levels were investigated. Basilar artery segments were investigated for cross-sectional area, wall thickness measurements and endothelial apoptosis. RESULTS: Telmisartan treatment restored the lowered level of Trx1, TrxR, TAS and the expression of TrxR1 seen in SAH. Telmisartan treatment also decreased TXNIP expression, TOS, MDA and TNF alpha levels. Morphological changes of cerebral vasospasm were attenuated after treatment. Endothelial apoptosis significantly reduced. DISCUSSION: Treatment with telmisartan ameliorates oxidative stress and SAH-induced cerebral vasospasm in rabbits. These effects of telmisartan may be associated with downregulation of TXNIP and upregulation of Trx/TrxR.

Treatment of Acute Vocal Fold Injury With Platelet-Rich Plasma.

OBJECTIVES: Platelet-rich plasma (PRP) is a reliable and has low side-effect profile and has beneficial effects on wound healing. Its investigatory effects on wound-healing process were shown on various tissues. The aim of the present study was to evaluate effectiveness of PRP application on scar tissue of acute vocal fold injury. MATERIALS AND METHODS: Twenty-four Wistar rats were used in the study. The entire layer of the lamina propria down to the thyroarytenoid muscle of 10 subjects was unilaterally injured by with a microscissor. Gelfoam-absorbed PRP was applied on the injured area for 10 minutes. Control group consisted of rats unilaterally injured using a microscissor, and gelfoam with normal saline was applied on the injured area. Following sacrifice, the larynxes were carefully dissected and removed for histopathologic examination. After excised larynx experiments, serial sections were prepared from vocal fold. Hematoxylin eosin and immunohistochemical staining were done for epithelial growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR1), and vascular endothelial growth factor (VEGF) staining for histopathologic examinations. RESULTS: There was not a significant difference between the two groups for lymphocyte. Although collagen and VEGF were higher in the study group, there was not a significant difference between the groups (P > 0.05). There was a significant difference between control and study groups for EGFR and FGFR1(P < 0.05). CONCLUSIONS: PRP has beneficial effects on wound healing. PRP accelerates epithelization of injured rat vocal folds by inducing EGFR secretion. PRP is an autogenous, reliable, low side-effect profile, easily harvested material. PRP may be useful to prevent scar formation.

Effect of Carvedilol on Secondary Damage in Experimental Spinal Cord Injury in Rats.

AIM: Previous studies have shown that carvedilol has a neuroprotective effect in animal models of brain ischemia and brain oxidative damage in vitro. This study was perfomed to investigate the effect of carvedilol on the secondary damage in experimental spinal cord injury (SCI). MATERIAL AND METHODS: Twenty-four Wistar albino rats were divided into three groups. Group 1 underwent laminectomy alone. Group 2 underwent laminectomy followed by SCI and received carvedilol. Group 3 underwent laminectomy followed by SCI and received no medication. The neurological functions were assessed by Tarlov's motor scale at the first and 24th hours. Oxidative stress status was assessed by MDA, SOD, MPO, GSH activities. A TUNEL-based apoptosis kit was used for evaluating apoptosis in the spinal cord samples and hematoxylinand eosin-stained specimens were used for light microscopic examination. RESULTS: Carvedilol reduced apoptosis and regulated oxidant and antioxidant status by increasing SOD and GSH levels and reducing MPO and MDA levels in the spinal tissue homogenate. Neurological examination of rats revealed statistically significant improvement 24 hours after the trauma. CONCLUSION: Carvedilol has a statistically significant therapeutic effect, especially on functional recovery, and we found that carvedilol reduced secondary damage by inhibiting apoptosis and regulating the oxidant and antioxidant status.

Common variable immunodeficiency and pulmonary amyloidosis: a case report.

Common variable immunodeficiency is the most common symptomatic primary immune deficiency characterized by hypogammaglobulinemia, recurrent infections, and increased risk of autoimmune disease and malignancy. Secondary amyloidosis develops from chronic inflammatory conditions. The co-existence of CVID (especially in patients with bronchiectasis) and secondary amyloidosis has been reported rarely. We describe the first case of pulmonary hypertension secondary to pulmonary amyloidosis in a patient with CVID.

High thioredoxin reductase 1 expression in meningiomas undergoing malignant progression.

Thioredoxin (Trx) is a redox active protein that regulates several physiological and biochemical functions, such as growth, apoptosis and cellular defense. The function of Trx itself is regulated by thioredoxin reductase (TrxR). This study was designed to determine the expression of TrxR1 in meningioma tissues of different World Health Organization grades (grade I-III). Meningioma tissues were extracted from the histopathological specimens of 29 patients. These samples included seven histologically normal meningeal tissues that served as a control group and 12 grade I, 12 grade II and 5 grade III meningioma samples. TrxR1 expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunostaining. The proliferative and apoptotic indices of the specimens were investigated by Ki-67 immunostaining and TUNEL assay, respectively. TrxR1 expression, as assessed by qRT-PCR, increased significantly with meningioma grade (p < 0.001). The immunostaining intensity of TrxR1 increased significantly with meningioma grade (p < 0.001). Ki-67 index values increased significantly in accordance with grade progression (p < 0.001). The apoptotic index values were not significantly different in any group (p > 0.05). Trx system seems to be involved in the malignant progression of meningiomas. Further, large studies are required to elucidate the exact role of this system.

The effect of dosage on the efficiency of LLLT in new bone formation at the expanded suture in rats.

The aim of this study was to investigate the effect that dosage has on the efficiency of low-level laser therapy (LLLT) in bone formation in a rat study model. Twenty-eight rats were divided into four groups as only expansion (OE), expansion + low dose (0.15 J) (LD), expansion + medium dose (0.65 J) (MD), and expansion + high dose (198 J) (HD) laser therapy groups. The midpalatal suture was expanded during 5 days. Afterwards, irradiations were started and performed with an 820 nm, continuous wave, Ga-Al-As diode laser (Doris, CTL-1106MX, Warsaw, Poland). At the end of experiment, the premaxillae of the animals were dissected. The sections were transferred into PC environment and analyzed by using Image Analysis program. Number of osteoblasts, osteoclasts, fibroblasts, vessels, transforming growth factor beta (TGF-ß) expression, and new bone formation were evaluated with this program. Amount of expansion did not show any difference among the groups. All parameters except the number of osteoclasts were increased in all lased groups while that parameter was significantly decreased. Vessels, TGF-ß expression, and new bone formation were mostly increased in LD group followed by HD group. Among the lased groups, a significant difference was observed only for the amount of new bone formation, which was between the LD and the MD groups. On the other hand, the difference in this parameter was insignificant between OE and MD groups. Low-level laser therapy with both 5 and 6,300 J/cm(2) doses was found to be significantly effective, while the 20 J/cm(2) dose did not show a significant effect in increasing new bone formation. This finding reveals that the efficiency of the therapy is affected by the dosage.

Tissue thioredoxin reductase-1 expression in astrocytomas of different grades.

Thioredoxin (Trx) is a redox active protein that regulates several physiological and biochemical functions, such as growth, apoptosis and cellular defense. The function of Trx itself is regulated by thioredoxin reductase (TrxR). Studies performed in a variety of human primary tumors have shown that thioredoxin reductase 1 (TrxR1) is overexpressed in tumoral tissues compared with corresponding normal tissues. This study was designed to determine the expression of TrxR1 in astrocytoma tissues of different World Health Organization (WHO) grades (grade I-IV). The proliferative (Ki-67) and apoptotic indices of the specimens were also investigated for correlation analysis. Astrocytoma tissues were extracted from the histopathological specimens of 40 patients. These samples included seven histologically normal brain tissues that served as a control group and ten tumoral samples for each grade of astrocytoma (grade I-IV). The histologically normal brain tissues were obtained from the non-tumoral portions of the pathological specimens of grade I (2 cases), grade II (2 cases), grade III (2 cases) and grade IV (1 case) astrocytomas. TrxR1 expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunostaining. The proliferative and apoptotic indices of the specimens were investigated by Ki-67 immunostaining and TUNEL assay, respectively. TrxR1 expression, as assessed by qRT-PCR, increased significantly with astrocytoma grade (p = 0.01). The immunostaining intensity of TrxR1 in grade IV astrocytomas was significantly greater than that in the control tissue and all other astrocytoma grades (p < 0.001). Similarly, immunostaining intensity of TrxR1 in the grade III astrocytomas was significantly greater than that in the control group and grade I astrocytomas (p < 0.001). All astrocytoma tissues showed more intense staining in ascending grades, but the differences between grade I and the control, grade II and the control, grades II and I, grades III and II were not statistically significant (p > 0.05). Ki-67 index values increased significant in accordance with grade progression (p = 0.01). The apoptotic index values were not significantly different in any group (p > 0.05); however, the differences between grade IV and the control and between grades IV and I were statistically significant (p < 0.05). Expression of TrxR1, as assessed by both qRT-PCR and immunostaining, correlated highly with both the astrocytoma grade and Ki-67 index.

Effects of diclofenac sodium and octreotide on treatment of caerulein-induced acute pancreatitis in mice.

BACKGROUND: Research continues to develop novel therapeutic modalities that particularly focus on the pathogenesis of acute pancreatitis. This study aimed to assess the effects of diclofenac sodium and octreotide, alone or in combination, on pancreatic enzymes, pancreatic myeloperoxidase activity, histopathology and apoptosis of pancreas cells, using a model of experimentally induced acute pancreatitis. OBJECTIVES: We aimed to demonstrate effects of diclofenac sodium, octreotide and their combined use on pancreatic enzymes, activity of pancreatic myeloperoxidase (MPO) activity, histopathology and apoptosis of pancreas on treatment of caerulin-induced experimental acute pancreatitis. MATERIALS AND METHODS: Caerulin-induced acute pancreatitis model was created using a total of 58 male BALB-C mice of 25 gr in seven groups. Serum amylase, lipase levels and pancreatic myeloperoxidase activity were examined as well as apoptotic values in pancreatic acinar cells through TUNNEL method. Histopathology of pancreas was evaluated for presence of edema, hemorrhage, parenchymal necrosis, fat necrosis, leukocyte infiltration, and fibrosis. RESULTS: In the diclofenac sodium group, apoptotic values in the pancreatic acinar cells were found to be statistically lower than in the acute pancreatitis group in terms of parenchymal necrosis and hemorrhage scores (P = 0.007, P = 0.002, and P = 0.052, respectively). No statistically significant differences were found in serum level of amylase, lipase, pancreatic myeloperoxidase activity and the other histopathological scores (P > 0.05). CONCLUSION: Diclofenac sodium, a cost-effective agent with a favorable side-effect profile, may represent a novel therapeutic agent for the treatment of acute pancreatitis. Findings of this study suggest a better efficacy for diclofenac sodium monotherapy as compared to octreotide alone or octreotide/diclofenac combination.

The biomechanical and histological effects of platelet-rich plasma on fracture healing.

PURPOSE: Platelet-rich plasma (PRP) includes growth factors and proteins that accelerate and stimulate bone regeneration and tissue recovery. The aim of this study was to evaluate the effects of PRP on fracture healing in terms of biomechanics and histology. METHODS: Seventy female rats were included in this experimental study. They were divided into three groups: Group I (no PRP, n = 30), Group II (PRP added, n = 30) and Group III (control, n = 10). The left femurs of the rats in Groups I and II were osteotomized and fixed by K-wires. Although no additional intervention was performed on Group I rats, PRP was applied to the fracture sites of Group II rats. The remaining ten rats were used as the control group of the biomechanical test (Group III). In the fourth week, nine femurs from Group I and ten femurs from Group II, and in the ninth week, nine femurs from each group were removed, and bone recovery was assessed histologically according to Modified Lane-Sandhu histological scoring criteria. Three-point bending test was applied to femurs for biomechanical evaluation in the ninth week. RESULTS: Histological healing was found to be significantly higher in Group II than in Group I (p < 0.05). Furthermore, biomechanical test results showed that healing quantity and bone strength were significantly better in Group II than in Group I (p < 0.05). CONCLUSION: PRP is a widely studied material in the physiology of fracture healing. The results of this study demonstrated the ameliorative biomechanical effects of PRP on fracture healing, in addition to accelerating the histological union of fractures. In the light of these results, PRP could be a viable alternative to accelerate the healing of fractures, late unions or non-unions. LEVEL OF EVIDENCE: Prospective comparative study, Level II.