Original Articles. Оncology
Purpose of the study. Was to evaluate overall survival of CC patients after D2 and D3 lymph node dissections and assess the frequency of postoperative complications and dynamics of postoperative recovery.
Materials and methods. The study included 50 CC patients aged 39–84 years.
Results. The comparative assessment of the overall survival, frequency of postoperative complications and dynamics of postoperative recovery was performed in patients divided into 2 groups depending on the lymph node dissection extent. Group 1 included 23 patients (46 %) with D2 lymph node dissection, group 2–27 patients (54 %) with D3 lymph node dissection. 8 (16 %) patients developed complications in the early postoperative period. Patients in group 1 had grade I (5 patients) and grade IIIb (1 patient) surgical complications, according to the Clavien-Dindo classification. 2 patients in group 2 developed grade III b complications. No significant differences were found between groups in peristalsis, gases and bowel movement, starting eating and drinking. A significant increase in the number of postoperative bed-days was observed in patients who underwent open surgery, compared with the group with laparoscopic access. No statistically significant difference was found in the overall survival in patients of the two groups (p = 0.918).
Conclusion. The rates of postoperative complications that required repeated surgery were comparable between the groups with D2 and D3 lymph node dissections. D3 lymph node dissection did not affect the dynamics of postoperative recovery and the overall survival of patients.
Purpose of the study. To substantiate the expediency of cellular immunotherapy in the treatment of patients with relapses of high-grade gliomas and evaluate the safety of injecting allogeneic cells directly into the cerebrospinal fluid.
Materials and methods. Our study included 5 patients, median age 7,6 years (2–16). Three patients had anaplastic astrocytoma (AA) (1st recurrence – 1 patient, 2nd recurrence – 2 patients), 1 patient had glioblastoma multiforme (GBM) (3rd recurrence) and 1 had diffuse brainstem glioma (BSG). The median time to the first relapse was 12 months (4 to 16), to the second one was 5 months (1 to 8). The protocol of immunotherapy included combined administration of autologous dendritic cell-based vaccine (DV) and repeated intrathecal/intraventricular injections of donor allogenic immunocompetent cells (alloIC) for at least 2 years.
Results. Two of 3 patients with AA experienced a progression-free interval of 67 and 71 months One patient with 3rd GBM relapse is alive without any therapy 13.3 years after immunotherapy start. The median time of follow-up was 67 months with the 2‑years overall survival was 58 %. Two patients died from disease progression within 6 and 7 months from the start of immunotherapy. Over the period of treatment the patients received a median of 20 (8 to 60) alloIC injections and 18 (8 to 44) DV administrations. No serious side-effect was observed.
Conclusion. Immunotherapy could be an attractive option for treating patients with high-grade malignant gliomas irresponsible to conventional therapy and is worthy of further investigation.
Purpose of the study. Evaluation of the antioxidant status and DNA damage in tissues of subcutaneous xenografts of non-small cell lung cancer and in peritumoral tissues created using the A549 and H1299 cell cultures.
Materials and methods. The study included 35 intact male Balb/c Nude immunodeficient mice. Cell lines A549 and H1299 were used as transplantable tumor biomaterial. A CDX model was created in accordance with the protocol for supratentorial injections (Ozawa T., James C. D., 2010) adapted for this experiment. Growth rates were controlled and intracranial xenografts were visualized using a high-resolution micro-CT system. The activity of catalase and superoxide dismutase was determined with non-denaturing electrophoresis in 8 % and 12 % polyacrylamide gel. The concentrations of sulfhydryl groups were determined according to Ellman. The DNA damage in lymphocytes was determined by the comet assay.
Results. The experiment resulted in the creation of models of brain tumors characterized by intracranial growth pattern in 100 %. The activity of catalase in the studied lysates of intracranial xenografts, peritumoral tissue and healthy tissues of tumor-bearing animals in all experimental groups increased statistically significantly relative to the healthy tissue of intact animals, and the greatest differences from the control were recorded in the group of animals with implanted H1299 culture at a concentration of 1 × 106 . Superoxide dismutase activity in the studied lysates of intracranial xenografts and peritumoral tissues statistically significantly increased compared to the control sample in all experimental groups. The highest increase in the SOD activity was observed in the tissues of intracranial xenografts with the highest tumor load, which amounted to 28.8 % and 32.9 % of the changes relative to the control sample. A statistically significant increase in the concentration of SH-groups relative to the control sample in tumor tissue lysates was revealed in all experimental groups, and the highest concentration (36.2 ± 0.47) was observed in the group of experimental animals with the highest tumor load. Percentage change in tail moment (DNA damage indicator) in groups O1, O2, O3 and O4 increased statistically significantly compared to the control sample by 55.8 %, 111.8 %, 97.3 % and 170 %, respectively.
Conclusions. The observed increase in the activity of the antioxidant defense system, accumulation of oxidative modifications of proteins, and an increase in DNA double-strand breaks in the tissues of intracranial xenografts of non-small cell lung cancer in vivo suggest that the created models reflect processes similar to those in tumors of human non-small cell lung cancer.
Purpose of the study. The investigation is aimed to provide a systematic comparison of different contrasting methods for in vivo micro-CT diagnostic of orthotopic colorectal cancer models extracted by ortotopic implantation into the caecum of immunocompromised mice BALB/c Nude lines.
Materials and methods. BALB/c Nude (N = 25) female mice were implanted by transplanted human colorectal cancer strain into the cecum. 20 days after the implantation mice were administered with iodine-based contrast agent Optiray by means of different administration method (intravenously, per os, intraperitoneally, per rectum) and micro-CT scans have been registered via Quantum GX2 tomograph. Measurement of tumor nodes was performed both by means of estimation from micro-CT images via RadiAnt DICOM Viewer software and by means of explicit measurements using calipers upon laparotomy and posthumously. At the last stage of the study, the animals were euthanized by cervical dislocation. The tumors were excised, measured with a caliper and placed in 10 % formalin for the standard histological analysis according to the standard methods.
Results. The average volumes of tumor xenografts in animals with intravenous, oral, and intraperitoneal contrast administration measured at micro-CT were 53.7 ± 5.2 mm3, 52.7 ± 6.4 mm3 and 63.6 ± 5.6 mm3 respectively; measured at laparotomy – 43.0 ± 5.5 mm3, 44.5 ± 5.4 mm3 and 58.5 ± 5.5 mm3 respectively; measured post-mortem – 55.2 ± 6.6 mm3, 53.2 ± 8.8 mm3 and 65.9 ± 3.8 mm3 respectively. The average volumes of tumor xenografts isolated post-mortem in these groups were comparable with the values shown at micro-CT, but larger than the volumes measured at laparotomy.
Conclusion. The results obtained demonstrated that intravenous, peroral and intraperitoneal administration techniques provide the best visualization of laboratory rodents pathological tissue upon in vivo micro-CT diagnostics and thus are preferred.
Purpose of the study. To study the levels of adrenal axis factors in the hypothalamus, adrenal glands, blood serum of mice and catecholamines in the adrenal glands during the independent growth of B16 melanoma and Lewis lung carcinoma (LLC) and their combined growth in female mice, and in males – with independent growth of B16 and combined growth of B16 and LLC.
Materials and methods. Male and female BALB/c Nude mice were divided into groups, n = 7 each: group 1 involved intact animals, group 2 involved mice with B16/F10 melanoma, group 3 – mice with LLC, group 4 – synchronous growth of melanoma and LLC. Levels of corticotropin releasing, noradrenaline and dopamine were determined in homogenates of the hypothalamus and adrenal glands and in the blood serum of all animals by ELISA, and levels of 17‑hydroxyprogesterone (17-OHP), dehydroepiandrosterone sulfate (DHEA-S) and cortisol were determined by RIA. Statistical processing of results was performed using the Statistica 10.0 program.
Results. All tumor-bearing females showed elevated corticotropin releasing in the hypothalamus together with an increase of all stress-characterizing parameters: cortisol, the cortisol/DHEA-S ratio, and noradrenaline. However, an increase in serum levels of cortisol was blocked by high levels of DHEA-S, and as a result, the cortisol/DHEA-S ratio was either within the normal range (B16 melanoma and B16+LLC combination) or reduced (LLC). Levels of corticotropin releasing in the hypothalamus of tumor-bearing males decreased, together with opposite changes in stress-characterizing parameters in the adrenal glands: cortisol increased, the cortisol/DHEA-S ratio did not differ significantly from the control values, and noradrenaline decreased. An increase in serum levels of cortisol was not blocked by high levels of DHEA-S, and as a result, the cortisol/DHEA-S ratio was sharply elevated in B16 melanoma and B16+LLC combination.
Conclusion. At independent and primary multiple types of tumor growth, the sex-specific features of the functioning of the adrenal axis at the central and peripheral levels are observed, which determines a more pronounced stressful state of the body with B16+LLC combination growth, realized by various mechanisms.
Purpose of the study. To evaluate the number of cells with the CD45+/- phenotype expressing Toll-like receptors (TLRs) in tissues of the tumor, peritumoral area and resection line tissues in colon cancer (CC) with various tumor locations.
Materials and methods. The study included 50 patients with CC. All patients underwent surgery as the primary treatment, and tissue material was collected from the patients. Expression of TLRs (2, 3, 4, 8, 9) on CD45+, CD45- cell populations was determined by flow cytometry in cell suspensions obtained from tissues of the tumor, peritumoral area (1–3 cm from the tumor) and resection line tissues (~10 cm from the tumor) with further calculation of the percentage of cells with the corresponding phenotype from the total number of cells.
Results. An analysis of left-sided colon tumors showed lower percentage of CD45- cells expressing TLR4, 8, compared to rightsided tumors, by 38 % and 25 %. A comparative analysis of the number of CD45+ cells expressing TLR 2, 4 showed their decrease by 81 % and 87 %, respectively, compared with right-sided tumors. An assessment of the data in the perifocal zone of left-sided colon tumors, compared with right-sided ones, demonstrated a decrease in the percentage of cells with the CD45- phenotype that express TLR4, by 61 %. Resection line tissues in left-sided tumors, compared with right-sided ones, showed a statistically significant increase in the percentage of CD45- cells that express TLR 2, 4 by 205 % and 55 %, respectively. The number of CD45+ cells expressing TLR 4 decreased by 87 %. An assessment of the number of cells expressing TLRs 3 and 9 in the tumor, peritumoral area and resection line tissues did not reveal significant differences.
Conclusions. Lower number of cells with CD45+ and CD45- phenotypes express TLRs 2, 4, 8 in left-sided colon tumors and their peritumoral tissues, compared to right-sided cancer.
Original Articles. Surgery
Purpose of the study. Development of criteria for evaluating the reaction of tissues in the implantation area after prosthetic repair of the hernia ring in an experiment using new digital image processing technologies.
Materials and methods. The experiment was carried out on 13 Wistar rats of different sexes. Under anesthesia, a 4 × 2 cm mesh prosthesis was implanted in the layer similar to the onlay epiaponeurotic. The preparation and staining of each preparation was carried out in two versions: with hematoxylin-eosin and according to Mallory on different glasses. The seasons for performing experiments are winter, summer. Microphotography was performed on a medical transmitted light microvisor Microvisor μVizo‑103 (AD LOMO, Russia) with a 3.2 Mpx matrix (full frame size 1024 × 882 px, infoband width 88 px) with Planachromat and Stigachromat lenses with a digital scale × 1 (i.e. no digital zoom). Microphoto processing was performed using the ImageJ 1.53k program (version for public use). The criterion for including a microphoto in the calculation of indicators was the presence of an ingrown implant fiber in the preparation or a cavity surrounding the implant.
Results. The zone adjacent to the implant, with a width of about 50 µm ("the zone of changes in adjacent tissues") significantly differs from the "zone of changes in distant tissues" by a more pronounced inflammatory reaction due to a larger absolute number of lymphocytes (р = 0.019 and fibroblasts (р = 0.017), giant cells of foreign bodies (р < 0.001), larger vascular area (р < 0.001) and extravasations (р = 0.002). The study of the "zone of changes in adjacent tissues" is of key importance in assessing the local response of the body to implantation. The key zones of the implantation area are identified: "the cavity surrounding the implant", "the zone of changes in adjacent tissues", "the zone of changes in distant tissues". The calculation of indicators within these zones allows you to get more detailed results when comparing study groups in the experiment.
Conclusion. An effective method for assessing the reaction of tissues in the implantation area after prosthetic hernia repair in an experiment using new digital imaging technologies that can be used using a conventional optical microscope and standard staining methods is proposed.
Purpose of the study. Study of the possibilities of pharmacological regulation of local and systemic inflammatory response, stimulation of reparative regeneration in prosthetic repair of abdominal wall hernias.
Materials and methods. The experiment was carried out on 92 rats of both sexes of a pure line, divided into 4 groups. Group I (control) received only 1 ml of 0.9 % sodium chloride solution. Group II – hydroxyethyldimethyldihydropyrimidine 21 mg/day. Group III – potassium orotate 10.5 mg/day. Group IV – methyluracil 14 mg/day. Under anesthesia a mesh implant 4 × 2 cm was placed in the layer between the hypodermis and the superficial fascia. Animals were withdrawn from the experiment on days 5 (n = 52) and 14 days (n = 40). The site of implantation for histomorphometric studies captured the dermis, hypodermis, implant layer, fascia, and muscle. The preparation and staining of the preparations were carried out with hematoxylin-eosin and by Mallory.
Results. 1. On the 5th day after the operation group II (hydroxyethyldimethyldihydropyrimidine) was characterized by pronounced neovasculogenesis (p = 0.002, U-test) and more active processes of formation and maturation of connective tissue (p = 0.024, U-test) with a low immune response to a foreign body (p = 0.044, U- test). III (potassium orotate) and IV groups (methyluracil) were characterized by a pronounced decrease in the inflammatory response with a simultaneous increase in tissue edema (p < 0.001, U-test), slowing down the formation and maturation of connective tissue (p = 0.016, U-test). 2. On the 14th day after the operation, group II (hydroxyethyldimethyldihydropyrimidine) was characterized an increase in the area of neovasculogenesis by 208.0 % (p < 0.001, U -criterion), a decrease in the volume of fluid accumulations by 63.4 % (p < 0.001, U-criterion), an increase in the processes of formation and maturation of connective tissue, mainly due to an increase in the number of fibrocytes by 333.3 % (p < 0.001, U-criterion). The parameters of the immune response to a foreign body had significant differences (p < 0.001, U-test).
Conclusion. The use of potassium orotate and methyluracil reduces the inflammatory response in the tissues of the implantation area, however, it is associated with the appearance of liquid components and with a decrease in the formation of connective tissue cells. The use of hydroxyethyldimethyldihydropyrimidine is devoid of the above disadvantages, it is characterized by the predominance of regeneration and neovasculogenesis processes, which allows it to be used as a pharmacological support drug for prosthetic hernioplasty.
Original Articles. Urology
Purpose of the study. The study’s objective is to investigate the influence of risk factors for recurrence of prostate cancer (PCa) after radical surgical treatment on the unfavourable course of recurrence of the disease with the formation of a set of the most significant factors of a model that reflects the likelihood of relapse.
Materials and methods. A retrospective analysis of clinical, pathomorphological and perioperative parameters of 803 patients with PCa after radical prostatectomy was carried out. By means of logistic regression, a model for assessing the risk of recurrence for patients with prostate cancer was built, which included 7 indicators, one of which was measured by quantity (time between biopsy and start of treatment) and six categorical ones (ISUP grade group, cT, cN+, positive surgical margin, PSA level after surgery, pN+). The construction of a logistic regression model consisted in obtaining a characteristic of the logistic function Ψ for the standard equation y = exp(ψ) / (1 + exp(ψ)).
Results. Substituting the coefficients obtained for each of the indicators, we obtain Ψ to assess the risk of relapse Ψ = 0.485 × X1+ 1.937 × X2 + 0.789 × X3 + 3.229 × X4 + 0.443 × X5 + 0.880 × X6 + 0.015 × X7–6.65. In the resulting formula, each of the regression coefficients describes the size of the contribution of the corresponding factor. In our case, all regression coefficients were positive, which means that this factor increases the overall risk of relapse. The quality of the resulting model is determined by the chi-square = 284.3; p < 0.001; OR = 28.45. The sensitivity of this model was 86.6 %, specificity 81.5 %, diagnostic accuracy 82.7 %.
Conclusion. This model makes it possible to obtain the probability of recurrence after radical prostatectomy depending on the severity of a specific set of predictive signs (a positive effect is predicted for y > 0.5, a negative one for y ≤ 0.5) and the degree of influence of one or a group of predictive signs on the likelihood of relapse, such as the ISUP grade group, locally advanced disease, clinically detectable lymph node lesion, positive surgical margin, PSA level of more than 0.09 ng/ml 1 month after surgery, the presence of regional metastases and the time between biopsy and the start of treatment.
Clinical Case Reports
Strokes account for 15–25 % of confirmed cases internal carotid artery (ICA) occlusion. The frequency of ICA occlusion in asymptomatic patients is unknown. The strategy of treatment patients with symptomatic ICA occlusion has not been determined. A clinical observation is presented with the stage of surgical treatment of a 67‑year-old patient with a developed stroke in the basin of the left middle cerebral artery. The examination according to MSCT angiography revealed occlusion of the left ICA. To assess the brain perfusion, a single-photon emission computed tomography (SPECT) with 99mTc was performed. Throb endarterectomy of the left internal carotid artery was performed, with a control ultrasound examination on the 6th day after the operation and after 6 months the blood flow through the internal carotid artery was preserved. Control SPECT with 99mTc was performed, in comparison with the previous study, a significant improvement perfusion on both hemispheres is determined. Improvements in the neurological status were noted in the form of a complete regression of neurological symptoms. On the sixth day after surgical treatment, the patient was discharged in a satisfactory condition. During the hospital stay, the patient was “examined” using a questionnaire for assessing the quality of life – The Short Form‑36 (SF‑36) and MoCA (Montreal Cognitive Impairment Assessment Scale). The survey was conducted on the 15th day of inpatient treatment, before surgery, and on the 6th day after surgery. After 6 months, the patient was re-interviewed. The results obtained indicate a correlation between the improvement of brain perfusion and cognitive impairment, as well as a positive effect of revascularization of the internal carotid artery on the patient’s quality of life. The presented results indicate not only the possibility, but also the high efficiency of surgical treatment of occlusive lesions of the internal carotid artery. Surgical treatment in the early stages after occlusion against the background of the collapse of atherosclerotic plaque and thrombosis of the lumen is a determining factor in the successful restoration of blood flow, improvement of brain perfusion.
It is reasonable to consider the technical possibility and oncological feasibility of the local tumor destruction in patients with locally advanced pancreatic cancer (PCa). Irreversible electroporation (IRE) is a non-thermal method of local tumor ablation, which uses non-thermal energy of high-voltage ultrashort electric fields localized between electrodes to create nanopores in the cellular wall with the following cellular death. The zone of impact can be accurately predicted using the location of the electrodes. A fairly clear and controlled ablation boundary without a clinically significant zone of perifocal tissue damage reduces the risk of accidental injury to the wall of a hollow organ. The method is based on a change in the permeability of the cell membrane and the development of apoptosis, which allows to act directly on the ducts and the great vessels infiltrated by the tumor without a high risk of damage. The presented case shows that IRE is advisable to use as a part of the combined treatment of patients with locally advanced PCa. There were no complications observed after the IRE. Radiological evaluations and pathologic reports showed an adequate long-term local control. Also, good results were obtained in the overall life expectancy, given that we are talking about unresectable ductal adenocarcinoma of the pancreas. The patient passed away in 39 months from the beginning of the treatment and in 26 months from the initial IRE. In case of local relapse, repeated electroporation with a good long-term result is also possible. The time to progression exceeded eleven months after electroporation performed for a local relapse. According to magnetic resonance imaging, both locoregional relapse and distant liver metastases were detected. The patient lived 16 months after a repeated IRE session and died of pulmonary embolism on the background of chemotherapy. Favorable prognostic factors are the presence of an objective response to previous conservative treatment, compliance with the parameters of electroporation, complete inclusion of tumor infiltration in the affected area.
Review
Russian and foreign epidemiological data suggest that males may have an increased risk of morbidity and mortality associated with COVID‑19. It is known that due to the high expression of ACE2 in spermatogonia, Leydig and Sertoli cells, the testicle, as an organ producing male germ cells, is a potential target for the COVID‑19 virus, which directly affects the reproductive health of men. To date, several factors of the influence of the SARS-CoV‑2 virus on the male reproductive system are known. To begin with, when infected, the virus has a direct damaging effect on the testicle itself with the provocation of orchitis. In addition to the previous statement, high body temperature during inflammation contributes to an increase in testicular temperature, which can lead to a deterioration in the quality of sperm. Furthermore, a pronounced inflammatory reaction promotes the circulation of a large number of cytokines and damage to the hemato-testicular barrier. Also, the increased activity of immunocytes leads to an imbalance of the antioxidant system of men, an increase in the level of reactive oxygen species and the formation of oxidative stress. In a Nutshell, Leydig cell dystrophy provokes the development of secondary hypogonadism. Thus, COVID‑19, like a “multifaceted Janus”, has a polysymptomatic manifestation, but at the same time has a multifactorial effect on the reproductive function of a man. All these factors of the impact of the SARS-CoV‑2 virus on the testicle must be considered when rehabilitating the patient. It should be kept in mind that the deterioration of sperm quality is observed both during the acute period of coronavirus infection and during recovery period. Therefore, laboratory evaluation of the ejaculate in dynamics and its correction is necessary for males, especially those who plan to realize their reproductive potential.
Conclusion. COVID‑19 generally has a negative impact on spermatogenesis and male fertility. To date, the timing and degree of restoration of a man’s reproductive function after a coronavirus infection remains completely unknown. Therefore, both in the early and late rehabilitation period, men of reproductive age need the supervision of an andrologist.
A few decades ago, colorectal cancer (CRC) was diagnosed much less frequently. It is currently the fourth and in some countries the third most lethal cancer in the world, with about one million deaths each year. Most colon tumors develop as a result of a multistep process involving a number of histological, morphological and genetic changes, the frequency of detection of which increases with the age of the patient. Timely screening aimed at identifying and removing precancerous neoplasms in the early stages can lead to a significant reduction in the incidence of CRC. Despite this, the currently diagnosed rate of CRC in everyday clinical practice is relatively low, however, it is predicted that with personalized, based on anamnesis, screening for CRC, the frequency of its detection will increase significantly. The practitioner needs to be aware of the risk factors leading to CRC and the various stages of disease progression in order to recommend appropriate screening strategies. The use of a surveys that includes risk factors in the collection of anamnesis will reduce economic costs and not miss a cohort of patients who need a detailed examination. The purpose of the review is to present current data on the epidemiology of CRC and risk factors contributing to the occurrence of CRC. A literature review of articles (original studies and reviews) published between 2016 and February 2022 on the study of risk factors for CRC was performed. The methods of bibliographic, informational and semantic search of sources in the databases of Google PubMed, Scopus were used. The article discusses in detail the epidemiology and highlights the modified and non-modified risk factors for CRC, including within different age categories. Data on early CRC in young people are presented in detail and carry clinical and molecular features, as well as the role of the hereditary factor in development.
Health Organization
Purpose of the study. Analysis of implemented personnel practices presented in dissertations in the specialty 14.02.03. Public health and healthcare for 2016–2020.
Materials and methods. We used data from the website of the Higher Attestation Commission under the Ministry of Science and Higher Education of the Russian Federation. Of the 220 dissertations, the authors of which were awarded the degree of candidate or doctor of medical sciences in the specialty 14.02.03 Public health and health care, 21 scientific papers devoted to personnel issues were singled out. In this study, a descriptive-analytical method was applied.
Results. Dissertations covering a fifteen-year period since the beginning of the modernization of healthcare revealed a number of problems: low wages; insufficient staffing, disproportion between doctors and paramedical workers; difficult working conditions, the outflow of personnel from the industry in general, as well as from public health institutions to private medical organizations. Approbation of developments and implementation on the basis of research, evaluation of their effectiveness allowed the authors to offer recommendations for implementation in healthcare practice, starting with the Ministry of Health of the Russian Federation, government bodies of the subject of the Federation, a medical organization, educational institutions and ending directly with the object of study – management personnel, doctors, secondary medical worker. The study and analysis of personnel practices presented in scientific research makes it possible to determine the most relevant areas for the formation of personnel policy.
Conclusion. The results of the study showed a small number of dissertations (9.5 %) devoted to personnel issues and defended in the specialty 14.02.03. Public health and healthcare, despite the decisive role of human resources in the innovative development of the country’s healthcare. The studies covered all groups of medical workers, managers, students of medical universities, colleges and included the study of the state of health, motivation, job satisfaction, leadership training, interaction between medical workers of different levels and patients. Effective practices are proposed that can be replicated at the regional level, new ones can be created on their basis, and they also make it possible to determine the criteria for assessing staff policy in a medical organization, subject and country.