Lysosomal enzymes in serum and synovial fluid in patients with osteoarthritis.

OBJECTIVE: To assess the activity of arylsulfatase (AS), acid phosphatase (ACP), cathepsin D (CAT-D) and alpha-1 antitrypsin (AAT) in blood serum and synovial fluid (SF) of patients with hip or knee osteoarthritis (OA). METHODS: The study included 43 subjects with OA (35 hip OA, 8 knee OA), submitted total joint replacement. The control group consisted of 58 subjects with no past history of musculoskeletal disorders. RESULTS: The OA blood serum samples showed a significantly higher level of lysosomal enzymes activity than in the control group (AS by 17.8%, AAT by 42.4%); only the CAT-D activity decreased by 50%). AS, ACP and CAT-D activities were about two-fold higher in SF when compared with blood of OA patients. The differences between the genders were visible in the SF: Total protein concentration, activity of ACP (both higher in OA men) and activity of CAT-D (higher in OA women). Between the involved hip and knee, there were no significant differences in all estimated parameters in the blood serum of the OA group. In regard to the SF, only ACP activity was significantly higher in patients with a hip involved. CONCLUSIONS: The osteoarthritic SF enzymatic profile differs from that in normal joints. The OA in joints is not reflected in the systemic response. Our preliminary results suggest further studies on role of lysosomal enzymes (ACP and AS) as biomarkers for the detection of osteoarthritis.

A new detection method for a newly revealed mechanism of pyrethroid resistance development in Varroa destructor.

The Varroa destructor mite has recently displayed an ever increasing resistance to new drugs, contributing to CCD proliferation. This work was aimed at determining new viable methods for identifying the pyrethroid resistance of V. destructor and DNA methylation in resistant and sensitive mites. DNA was extracted from Varroa mites. Nucleotide changes in the DNA of pyrethroid-resistant, pyrethroid-sensitive, and control mites were identified with polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in the case of five mitochondrial gene fragments. More bands were observed in the drug-resistant mites than in the other two groups. Sequencing confirmed these observations. Decreased global DNA methylation levels were observed in the pyrethroid-resistant mites. There exists a previously undescribed mechanism of pyrethroid resistance development in Varroa mites. The PCR-SSCP methods can be considered and further developed as useful tools for detecting V. destructor resistance.

Coenzyme Q10 treatments influence the lifespan and key biochemical resistance systems in the honeybee, Apis mellifera.

Natural bioactive preparations that will boost apian resistance, aid body detoxification, or fight crucial bee diseases are in demand. Therefore, we examined the influence of coenzyme Q10 (CoQ10, 2,3-dimethoxy, 5-methyl, 6-decaprenyl benzoquinone) treatment on honeybee lifespan, Nosema resistance, the activity/concentration of antioxidants, proteases and protease inhibitors, and biomarkers. CoQ10 slows age-related metabolic processes. Workers that consumed CoQ10 lived longer than untreated controls and were less infested with Nosema spp. Relative to controls, the CoQ10-treated workers had higher protein concentrations that increased with age but then they decreased in older bees. CoQ10 treatments increased the activities of antioxidant enzymes (superoxide dismutase, GPx, catalase, glutathione S-transferase), protease inhibitors, biomarkers (aspartate aminotransferase, alkaline phosphatase, alanine aminotransferase), the total antioxidant potential level, and concentrations of uric acid and creatinine. The activities of acidic, neutral, and alkaline proteases, and concentrations of albumin and urea were lower in the bees that were administered CoQ10. CoQ10 could be taken into consideration as a natural diet supplement in early spring before pollen sources become available in the temperate Central European climate. A response to CoQ10 administration that is similar to mammals supports our view that Apis mellifera is a model organism for biochemical gerontology.

The Morphological Image of Fat Body and Tergal Gland Cells in Uninseminated Apis mellifera Queen Bees.

The morphological changes in fat body cells, tergal gland cells, and the surface areas of the cell nuclei were determined in queen bees of the subspecies Apis mellifera carnica. This study focused on 1-, 8-, and 20-day-old uninseminated females kept in colonies, analyzing cells from three locations in the abdomen: the sternite, and tergites III and V. The oenocytes in the sternites were large, oval/circular with a centrally located nucleus, while in tergites III and V, they were small and triangular in the 1-day-old queens. During the first week of life, these cells in tergites III and V change their shape to oval and increase their sizes. The initially light yellow and then dark yellow granularities in the oenocytes of the fat body appear along with the advancing age of the queens. The trophocytes (sternites, tergites III and V) in the 1-day-old queens were completely filled with droplets of different sizes. In the 8- and 20-day-old queens, the number and size of the droplets decreased in the trophocytes of tergites III and V. The tergal gland cells had a centrally located cell nucleus in the 1-, 8- and 20-day-old queens. The dark granularities in these cells were visible only in the 20-day-old queens. Different morphological images of the fat body at the sternite, and tergites III and V, and the difference in the size of the oenocyte cell nuclei may indicate various functions of the fat body depending on its location. Characterization of the changes in the morphology of the fat body, taking into account its segmental character, and the tergal glands requires further research in older queens, e.g., one-year-old, brooding queens.

Effect of Rearing in Small-Cell Combs on Activities of Catalase and Superoxide Dismutase and Total Antioxidant Capacity in the Hemolymph of Apis mellifera Workers.

Honeybee nests constructed without man-made wax foundation have significantly more variability of cell widths/sizes than those in commercially-kept colonies. The effects of this natural variability in comb cell widths on individual and colony traits have not been explained to date. The investigation of this problem can lead to new findings about the biology, physiology, and possibly, the evolution of the honeybee. The aim of the study was to compare the catalase and superoxide dismutase activities and the total antioxidant capacity levels in the hemolymph of honeybee workers reared in small-cell combs and standard-cell combs in colonies kept simultaneously on standard- and small-cell combs. The ratio of the small-cell combs to the standard-cell combs in the nest was 1:1. The workers reared in small-cell combs were characterized by higher antioxidant activities in the hemolymph than those reared in standard-cell combs. Consequently, their hemolymph had a greater antioxidant capacity, which indicates that they may be better predisposed to be foragers than workers reared in standard-cell combs. To describe the physiological differences between worker bees reared in small- and standard-cell combs in the same colony, the role of the considerable variation in the cell width in natural combs built without the use of artificially produced wax foundation is worth elucidating. The comparison of the apiary and cage experiments indicated that changes in antioxidant activities predominantly result from worker activities, especially those requiring the intensification of metabolism, rather than the age of the worker bees. To reduce the impact on the results of random environmental factors potentially present in one-season studies of honeybee research, investigations should preferably be carried out over a few consecutive years.

Workers of Apis mellifera Reared in Small-Cell Combs Show Higher Activity of the Proteolytic System in Hemolymph than Workers Reared in Standard-Cell Combs in Laboratory Cage Tests.

The aim of the study was to compare the activities of proteases and their inhibitors in the hemolymph of honeybee workers reared in small-cell combs (SMC) and standard-cell combs (STC) in laboratory cage tests. The analyses conducted in laboratory conditions facilitated assessment of the impact of the comb cell width (small vs. standard) along with eliminating the influence of many environmental factors on the results. The width of the comb cells in which the workers were reared had a significant effect on the protein concentrations and proteolytic system activities in the hemolymph. Irrespective of the age of the workers, higher protein concentrations were found in the hemolymph of the SMC workers. In turn, the activities of proteases and their inhibitors in the hemolymph of 1-day-old bees were higher in the STC workers. In older bees, aged 7-21 days, activity was higher in the SMC workers. The role of the considerable cell width variability in natural combs that were built without the use of an artificially produced wax foundation is worth investigating. It is highly probable that the impact of the comb cell width on the features of workers reared in these combs modifies the age polyethism in the worker caste as well. The investigation results of one-season studies of honeybees could be seriously affected by random factors. To reduce the risk of these effects, it is advisable to continue experiments over a few consecutive years.

Raised jugular venous pressure intensifies release of brain injury biomarkers in patients undergoing cardiac surgery.

OBJECTIVES: Neurologic damage after cardiac surgery with extracorporeal circulation is multifactorial. Despite several studies, its pathophysiology is poorly understood. The purpose of this study was to determine the changes in jugular venous pressure and to analyze their effect on perioperative brain injury measured by biomarkers in patients undergoing coronary artery bypass grafting. DESIGN: Observational study. SETTING: Department of cardiac surgery in a medical university hospital. PARTICIPANTS: Adult patients undergoing elective coronary artery bypass grafting with extracorporeal circulation under general anesthesia. INTERVENTIONS: The right jugular vein was cannulated in retrograde fashion. Jugular venous pressure was measured in the jugular vein bulb (JVBP). Concentrations of plasma glial fibrillary acidic protein, tau protein, arteriovenous lactate, and jugular vein saturation were measured as the markers of brain injury during the surgery and early postoperative period. All were analyzed in relation to JVBP. MEASUREMENTS AND MAIN RESULTS: Increased JVBP was noted after extracorporeal circulation and after surgery. A significant increase >12 mmHg for JVBP, increased plasma glial fibrillary acidic protein, tau protein, arteriovenous lactate concentrations, and decreased jugular vein saturation were observed. CONCLUSIONS: Cardiac surgery increased JVBP and an increased JVBP > 12 mmHg intensified an increase in brain injury biomarker concentrations.

Effect of Comb Cell Width on the Activity of the Proteolytic System in the Hemolymph of Apis mellifera Workers.

This study is a continuation of the innovative research of the impact of rearing of bee colonies simultaneously on standard- and small-cell combs on the traits of worker bees and bee colonies. Its aim was to compare the activities of proteases and their inhibitors in the hemolymph of workers reared in a small-cell comb (SMC) and a standard-cell comb (STC) in colonies kept simultaneously on standard- and small-cell combs. The width of comb cells in which workers are reared has a significant effect on the protein concentration and the proteolytic system in the hemolymph, which is reflected in the activities of proteases and their inhibitors. The protein concentrations in the 1-day-old workers were always higher (p ≤ 0.05) in the SMC than STC workers. The opposite was found in the older bee workers (aged 7, 14 and 21 d). The activities of proteases and their inhibitors in the 1-day-old workers were always higher (usually significantly at p ≤ 0.05) in STC than SMC workers, and opposite results were observed in the groups of the older workers (aged 7, 14 and 21 d). The differences between the workers from small-cell combs and those reared in standard-cell combs may be related to their different tasks. Workers reared in small-cell combs probably work as foragers outside the nest, whereas bees reared in standard-cell combs work in the nest. This hypothesis requires confirmation. To reduce the impact of accidental determinants on the results of single-season research on honeybees, it is advisable that such investigations should be conducted for several consecutive years.

Dead Brood of Apis mellifera Is Removed More Effectively from Small-Cell Combs Than from Standard-Cell Combs.

The efficiency of the hygienic behaviour in bee colonies towards dead brood was assessed in small-cell combs (SMCombs) and in standard-cell combs (STCombs). Each colony had both types of combs in the nest on a permanent basis. Simultaneous keeping of a colony on standard- and small-cell combs is a novel approach to the use of small-cell combs in beekeeping. The number of killed pupae removed within 24 h was the measure of the hygienic behaviour efficiency. Regardless of the year, the brood in the SMCombs was uncapped and removed significantly more efficient (p ≤ 0.01) than in the STCombs (number of non-uncapped cells: in 2020 SMCombs = 3.79, STCombs = 11.62; in 2021 SMCombs = 2.34, STCombs = 5.28 and completely removed cells: in 2020 SMCombs = 87.46, STCombs = 80.04; in 2021 SMCombs = 96.75, STCombs = 92.66). In colonies kept simultaneously on standard- and small-cell combs, the width of the comb cells has a significant effect on the efficiency of removal of dead brood, which is removed more efficient from small-cell combs than from standard-cell combs.

Activities of Antioxidant and Proteolytic Systems and Biomarkers in the Fat Body and Hemolymph of Young Apis mellifera Females.

The proteolytic and antioxidant systems are important components of humoral immunity, and these biomarkers indicate the immune status. These compounds are synthesized in the bees' fat body and released into the hemolymph. Their functions maintain the organism's homeostasis and protect it against adverse environmental factors (including pathogens). We determined the activities of acidic, neutral, and alkaline proteases and their inhibitors, as well as superoxide dismutase (SOD), catalase (CAT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and the level of total antioxidant potential (TAC). These compounds were investigated in the fat body and hemolymph in the females with increased reproductive potential, i.e., queens and rebels, and in normal (non-reproductive sterile non-rebel) workers. The phenoloxidase (PO) activities were determined in the hemolymph. The normal workers had higher activities of proteases and their inhibitors, SOD and CAT, in the fat body and hemolymph, compared to the queens and rebels. The protease inhibitors were not usually active in the queens. As we predicted, the rebels revealed values between those of the queens and normal workers. The highest activities of proteases and antioxidants were identified in the fat body from the third tergite in comparison with the sternite and the fifth tergite. These results are important for oxidative stress ecology and give a better understanding of the functioning of the fat body and the division of labor in social insects.

Morphology of Nasonov and Tergal Glands in Apis mellifera Rebels.

Social insect societies are characterized by a high level of organization. This is made possible through a remarkably complex array of pheromonal signals produced by all members of the colony. The queen's pheromones signal the presence of a fertile female and induce daughter workers to remain sterile. However, the lack of the queen mandibular pheromone leads to the emergence of rebels, i.e., workers with increased reproductive potential. We suggested that the rebels would have developed tergal glands and reduced Nasonov glands, much like the queen but contrary to normal workers. Our guess turned out to be correct and may suggest that the rebels are more queen-like than previously thought. The tergal gland cells found in the rebels were numerous but they did not adhere as closely to one another as they did in queens. In the rebels, the number of Nasonov gland cells was very limited (from 38 to 53) and there were fat body trophocytes between the glandular cells. The diameters of the Nasonov gland cell nuclei were smaller in the rebels than in the normal workers. These results are important for understanding the formation of the different castes of Apis mellifera females, as well as the division of labor in social insect societies.

The concentration of thiobarbituric acid reactive substances (TBARS) and paraoxonase activity in blood of patients with osteoarthrosis after endoprosthesis implantation.

BACKGROUND: The aim of this study was to evaluate the concentration of malondialdehyde (MDA) in erythrocytes and in blood plasma and the activity of blood paraoxonase (PON1) of patients with osteoarthrosis (OA) submitted to endoprosthesis implantation for evaluating oxidative stress. MATERIAL/METHODS: Study was conducted on 55 patients with OA and on 54 total movement-efficient volunteers. The material for the study was venous blood plasma, serum and erythrocytes. RESULTS: Increased concentration of MDAe before surgery was observed in the group of men and in patients with a degenerative process affecting hip joints. After an implantation of endoprosthesis, MDAe decreased to the level observed in the control groups. In the study group MDA concentration in plasma was slightly lower before surgery, and after an operation it reached the value of the parameter of the reference groups. Regardless of sex or age, paraoxonase activity was almost twice as high in almost all subgroups as in the reference group. A positive correlation between PON 1 activity and MDAe concentration was demonstrated both before and after surgery in the group of men. CONCLUSIONS: The increase of PON1 activity in patients' serum in relation to the control groups indicates a probable pathogenic role of the increased formation of reactive oxygen species in the course of OA and may suggest acute inflammation of the synovial joint. The high level of PON 1 activity after endoprosthesis implantation indicates that surgical treatment may additionally stimulate ROS generation. MDAe concentration indicate more intensive process of lipid peroxidation in the elderly.

Reproductive Potential Accelerates Preimaginal Development of Rebel Workers in Apis mellifera.

Rebel workers develop from eggs laid by the previous queen, before it went swarming and left the colony orphaned, until the emergence of a new queen. In contrast to normal workers developing in the queen's presence, rebels are set to reproduce and avoid rearing of successive bee generations. They have more ovarioles in their ovaries, as well as more developed mandibular glands and underdeveloped hypopharyngeal glands, just like the queen. We posited that rebels are not only similar to queens in some anatomical features, but also develop in a shorter time in comparison to normal workers. Therefore, the aim of this study was to compare preimaginal development duration in rebel and normal workers. The results show that rebels, i.e., workers with a higher reproductive potential, had a significantly shorter preimaginal development period (mean ± SD, 19.24 ± 0.07 days) than normal workers (22.29 ± 0.32 days). Our result confirmed that workers who develop in a queen-less colony undergo a shorter preimaginal development than those in a queen-right colony.

Segmentation of the subcuticular fat body in Apis mellifera females with different reproductive potentials.

Evolution has created different castes of females in eusocial haplodiploids. The difference between them lies in their functions and vulnerability but above all in their reproductive potentials. Honeybee queens are highly fertile. On the other hand, the workers are facultatively sterile. However, rebel workers, i.e. workers that develop in a queenless colony, reproduce more often than normal workers. As a result, the fat body of these bees, which apart from acting as the energy reserve, is also the site of numerous metabolic processes, had to specialize in different functions perfected over millions of years of eusocial evolution. Assuming that the variety of functions manifests itself in the pleomorphic structure of the fat body cells, we predicted that also different parts of the fat body, e.g. from different segments of the abdomen, contain different sets of cells. Such differences could be expected between queens, rebels and normal workers, i.e. females with dramatically different reproductive potentials. We confirmed all these expectations. Although all bees had the same types of cells, their proportion and segmental character corresponded with the caste reproductive potential and physiological characteristics shaped in the evolutionary process. The females with an increased reproductive potential were characterized by the presence of oenocytes in the third tergite and high concentrations of compounds responsible for energy reserves, like glucose, glycogen and triglycerides. Queens had very large trophocytes, especially in the third tergite. Only in workers did we observe intercellular spaces in all the segments of the fat body, as well as high protein concentrations-especially in the sternite. As expected, the rebels combined many features of the queens and normal workers, what with other findings can help understand the ways that led to the origin of different castes in females of eusocial Hymenoptera.

Oxidative equilibrium in the prophylaxis of degenerative joint changes: an analysis of pre- and postoperative activity of antioxidant enzymes in patients with hip and knee osteoarthritis.

BACKGROUND: Osteoarthritis is often accompanied by disturbance of oxidative equilibrium. The aim of the study was to analyze antioxidant defense system function in patients with hip and knee osteoarthritis by assessing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity in erythrocytes. MATERIAL/METHODS: The study included 26 women and 18 men who underwent hip or knee arthroplasty. The pre- and postoperative activities of the antioxidant enzymes were evaluated. SOD activity was determined using the Misra/Fridovich method, CAT by the Beer/Seizer method, and the activity of GPx by the Paglia/Valentine method. The results were compared with those of a healthy control group. RESULTS: SOD activity of the patients was insignificantly lower than in the control group. However, after arthroplasty it increased in subgroups: in males by 13% (p<0.04), in females by 7% (p<0.05), in the group <69 years of age by ca. 14%) (p<0.01), and in the group with idiopathic OA by 12% (p<0.005). The activities of CAT and GPx in all the patient groups were significantly higher than in the controls. It increased even more on the 10th day after arthroplasty with the exception of the patients with rheumatoid osteoarthritis, in whom a decrease by 65.5% (p<0.008) were found, and in the patients > or =69 years old by 9.3% (NS). CONCLUSIONS: Knowledge of antioxidant enzyme activity might offer new targets for future therapeutic methods for the treatment of degenerative joint disease.

Toxin A-producing Clostridium difficile as an aetiological factor of post-traumatic wound infection.

Clostridium difficile is a well-known cause of hospital-acquired infection such as antibiotic associated diarrhoea or pseudomembranous colitis. Extraintestinal infections caused by this pathogen are described rarely. A case of post-traumatic wound infection caused by C. difficile in an immunocompetent, young and otherwise healthy trauma patient is reported. A 31-year-old female, a car accident victim, was admitted to hospital because of polytrauma. After open reduction and internal fixation of a supracondylar femoral fracture by means of the dynamic condylar screw (DCS) system, a purulent fistula occurred. Microbiological examination of the pus revealed C. difficile as the single aetiological factor of this infection. Empirical antibiotic treatment with cefazoline and metronidazole had been administered right after the surgery, but was found to be ineffective. The strain isolated from the patient was sensitive to most antimicrobials except for clindamycin, and amoxicillin/clavulanic acid was chosen for the guided therapy. Such treatment combined with the removal of the DCS system produced a desirable effect.

Evaluation of Selected Parameters of Lipid Peroxidation and Paraoxonase Activity in Blood of Patients with Joint Osteoarthritis.

BACKGROUND: Oxidative stress constitutes an important risk factor for tissue/cell damage and is involved in the occurrence and progression of Osteoarthritis (OA). OBJECTIVE: The aim of this study was to evaluate the concentration of Malondialdehyde (MDA) in Synovial Fluid (SF) and blood as well as the activity of serum and SF paraoxonase (PON1) of patients with OA. Our study aimed to assess local and systemic responses to oxidative stress. METHODS: The study included 43 subjects with OA qualified to total joint replacement. The control group consisted of 58 subjects with no musculoskeletal disorders. The malondialdehyde level were measured in venous blood plasma, erythrocytes (MDAe) and Synovial Fluid (SF) while PON1 activities were estimated in serum and SF. RESULTS: No differences in MDA concentrations in erythrocytes and blood plasma between healthy subjects and OA patients were observed. The significant higher level of MDA in SF compared with blood serum was seen in both genders (in woman about 46.2%, in men - 75.0%) and in both assessed joints (in hip about 53.8%, in knee - 115.9%). Total protein concentration and PON1 activity in serum were significantly higher in OA patients (about 14.6% and 87.9% respectively). The systemic response to osteoarthritis (serum PON1 activity) was higher about 33.6% in men than women and not dependent on affected joint. CONCLUSION: The changes observed in osteoarthritic SF show that the process of antioxidant enzymatic defence is intensified locally, not systemically. The lipid hydroperoxides elimination may lead to restore joints homeostasis.

A New Method for Quick and Easy Hemolymph Collection from Apidae Adults.

Bio-analysis of insects is increasingly dependent on highly sensitive methods that require high quality biological material, such as hemolymph. However, it is difficult to collect fresh and uncontaminated hemolymph from adult bees since they are very active and have the potential to sting, and because hemolymph is rapidly melanized. Here we aimed to develop and test a quick and easy method for sterile and contamination-free hemolymph sampling from adult Apidae. Our novel antennae method for hemolymph sampling (AMHS), entailed the detachment of an antenna, followed by application of delicate pressure to the bee's abdomen. This resulted in the appearance of a drop of hemolymph at the base of the detached antenna, which was then aspirated using an automatic pipetter. Larger insect size corresponded to easier and faster hemolymph sampling, and to a greater sample volume. We obtained 80-100 µL of sterile non-melanized hemolymph in 1 minute from one Bombus terrestris worker, in 6 minutes from 10 Apis mellifera workers, and in 15 minutes from 18 Apis cerana workers (+/-0.5 minutes). Compared to the most popular method of hemolymph collection, in which hemolymph is sampled by puncturing the dorsal sinus of the thorax with a capillary (TCHS), significantly fewer bees were required to collect 80-100 µL hemolymph using our novel AMHS method. Moreover, the time required for hemolymph collection was significantly shorter using the AMHS compared to the TCHS, which protects the acquired hemolymph against melanization, thus providing the highest quality material for biological analysis.