The treatment of aneurysmal bone cysts.

PURPOSE OF REVIEW: Aneurysmal bone cysts are rare, locally aggressive bone tumors. Optimal treatment of ABCs is still matter of debate as therapies including sclerotherapy, selective arterial embolization and systemic treatment with denosumab are increasingly utilized, in addition to or instead of traditional curettage. The purpose of this review is to discuss current concepts and difficulties in diagnosing and treating primary ABCs, based on latest available literature. RECENT FINDINGS: In diagnostics, multiple new fusion partners of USP-6 have been described on next-generation sequencing specifically for primary ABCs. In a recent systematic review, failure rates of percutaneous injections and surgery were comparable. In a literature review, the use of denosumab seemed effective but resulted in multiple cases of severe hypercalcemia in children. SUMMARY: Accurately diagnosing primary ABC is crucial for treatment decisions. Curettage remains a valid treatment option, especially with adjuvant burring, autogenous bone grafting and phenolization. Percutaneous sclerotherapy represents a solid alternative to surgery, with polidocanol showing good results in larger studies. Systematic therapy with denosumab exhibits favorable results but should be reserved in the pediatric population for unresectable lesions, as it may result in severe hypercalcemia in children. When selecting a treatment option, localization, stability and safety should be considered.

Do's and Don'ts in Primary Aneurysmal Bone Cysts of the Proximal Femur in Children and Adolescents: Retrospective Multicenter EPOS Study of 79 Patients.

BACKGROUND: Aneurysmal bone cysts (ABC) are rare benign cystic bone tumors, generally diagnosed in children and adolescents. Proximal femoral ABCs may require specific treatment strategies because of an increased pathologic fracture risk. As few reports are published on ABCs, specifically for this localization, consensus regarding optimal treatment is lacking. We present a large retrospective study on the treatment of pediatric proximal femoral ABCs. METHODS: All eligible pediatric patients with proximal femoral ABC were included, from 11 tertiary referral centers for musculo-skeletal oncology (2000-2021). Patient demographics, diagnostics, treatments, and complications were evaluated. Index procedures were categorized as percutaneous/open procedures and osteosynthesis alone. Primary outcomes were: time until full weight-bearing and failure-free survival. Failure was defined as open procedure after primary surgery, >3 percutaneous procedures, recurrence, and/or fracture. Risk factors for failure were evaluated. RESULTS: Seventy-nine patients with ABC were included [mean age, 10.2 (±SD4.0) y, n=56 male]. The median follow-up was 5.1 years (interquartile ranges=2.5 to 8.8).Index procedure was percutaneous procedure (n=22), open procedure (n=35), or osteosynthesis alone (n=22). The median time until full weight-bearing was 13 weeks [95% confidence interval (CI)=7.9-18.1] for open procedures, 9 weeks (95% CI=1.4-16.6) for percutaneous, and 6 weeks (95% CI=4.3-7.7) for osteosynthesis alone ( P =0.1). Failure rates were 41%, 43%, and 36%, respectively. Overall, 2 and 5-year failure-free survival was 69.6% (95% CI=59.2-80.0) and 54.5% (95% CI=41.6-67.4), respectively. Risk factors associated with failure were age younger than 10 years [hazard ratios (HR)=2.9, 95% CI=1.4-5.8], cyst volume >55 cm 3 (HR=1.7, 95% CI=0.8-2.5), and fracture at diagnosis (HR=1.4, 95% CI=0.7-3.3). CONCLUSIONS: As both open and percutaneous procedures along with osteosynthesis alone seem viable treatment options in this weight-bearing location, optimal treatment for proximal femoral ABCs remains unclear. The aim of the treatment was to achieve local cyst control while minimizing complications and ensuring that children can continue their normal activities as soon as possible. A personalized balance should be maintained between undertreatment, with potentially higher risks of pathologic fractures, prolonged periods of partial weight-bearing, or recurrences, versus overtreatment with large surgical procedures, and associated risks. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Altered CD4+ T cell immunity in nurses occupationally exposed to viral pathogens.

Pathogen exposure, including but not limited to herpesviruses, moulds the shape of the immune system, both at a basal state and in response to immune challenge. However, little is known about the impact of high exposure to other viruses on baseline immune signatures and how the immune system copes with repetitive exposures to maintain a balanced functionality. Here we investigated baseline immune signatures, including detailed T cell phenotyping, antigen-specific CD4+ and CD8+ T cell responses and cytokine profile in paediatric (PED) nurses, who have high occupational exposure to viral pathogens including varicella zoster virus (VZV) and respiratory viruses, and in neonatal intensive care unit (NICU) nurses, as a control group with infrequent occupational exposure. Our results show a lower CD4+ T cell response to two VZV proteins (IE62 and gE) and to tetanus toxoid (TT) in PED nurses who are cytomegalovirus (CMV)-seronegative, compared to CMV-seronegative NICU nurses, and that the decline might be more pronounced the more sustained the exposure. This decline might be due to an attrition of VZV- and TT-specific T cells as a result of the continuous pressure on the CD4+ T cell compartment. Moreover, our data suggest that the distinct T cell phenotypes known to be associated with CMV-seropositivity might be less prominent in PED nurses compared to NICU nurses, implying a plausible attenuating effect of occupational exposure on CMV-associated immunosenescence. Overall, this pilot study reveals an impact of occupational exposure to viral pathogens on CD4+ T cell immunity and supports further investigation in a larger cohort.

Distinct in vitro Complement Activation by Various Intravenous Iron Preparations.

BACKGROUND: Intravenous (IV) iron preparations are widely used in the treatment of anemia in patients undergoing hemodialysis (HD). All IV iron preparations carry a risk of causing hypersensitivity reactions. However, the pathophysiological mechanism is poorly understood. We hypothesize that a relevant number of these reactions are mediated by complement activation, resulting in a pseudo-anaphylactic clinical picture known as complement activation-related pseudo allergy (CARPA). METHODS: First, the in-vitro complement-activating capacity was determined for 5 commonly used IV iron preparations using functional complement assays for the 3 pathways. Additionally, the preparations were tested in an ex-vivo model using the whole blood of healthy volunteers and HD patients. Lastly, in-vivo complement activation was tested for one preparation in HD patients. RESULTS: In the in-vitro assays, iron dextran, and ferric carboxymaltose caused complement activation, which was only possible under alternative pathway conditions. Iron sucrose may interact with complement proteins, but did not activate complement in-vitro. In the ex-vivo assay, iron dextran significantly induced complement activation in the blood of healthy volunteers and HD patients. Furthermore, in the ex-vivo assay, ferric carboxymaltose and iron sucrose only caused significant complement activation in the blood of HD patients. No in-vitro or ex-vivo complement activation was found for ferumoxytol and iron isomaltoside. IV iron therapy with ferric carboxymaltose in HD patients did not lead to significant in-vivo complement activation. CONCLUSION: This study provides evidence that iron dextran and ferric carboxymaltose have complement-activating capacities in-vitro, and hypersensitivity reactions to these drugs could be CARPA-mediated.

Retinol binding protein and vitamin D associations with serum antibody isotypes, serum influenza virus-specific neutralizing activities and airway cytokine profiles.

Vitamin A supports the induction of immunoglobulin (Ig)A responses at mucosal surfaces in mice, but much less is known about the influence of vitamins on antibody isotype expression in humans. To address this knowledge gap, we examined 46 residual blood samples from adults and children, some of whom were experiencing influenza virus infections of the respiratory tract. Assays were performed for retinol binding protein (RBP, a surrogate for vitamin A), vitamin D (a related vitamin) and antibody isotypes. Results showed that all but two tested samples exhibited RBP and/or vitamin D insufficiencies or deficiencies. Vitamin D correlated with blood IgM and IgG3, while RBP correlated with IgG4 and IgA. RBP also correlated positively with age and with influenza virus-specific antibody neutralization titres. Individuals with low blood RBP levels exhibited the highest frequencies of over-expressed cytokines and growth factors in nasal wash samples, an indication of inflamed mucosal tissues. While cause-effect relationships were not discerned, results support a hypothesis that vitamins directly influence B cell isotype expression in humans, and by so doing may help protect mucosal surfaces from respiratory viral disease.

Risk assessment and management of Chlamydia psittaci in poultry processing plants.

Chlamydia psittaci causes respiratory disease in poultry and can be transmitted to humans. Historical outbreaks of psittacosis in poultry workers indicated the need for higher awareness and an efficient risk assessment and management. This group reviewed relevant previous research, practical guidelines, and European directives. Subsequently, basic suggestions were made on how to assess and manage the risk of psittacosis in poultry processing plants based on a classical four-step approach. Collective and personal protective measures as well as the role of occupational medicine are described. Despite the finding that exposure is found in every branch, abattoir workstations seem to be associated with the highest prevalence of psittacosis. Complete eradication is difficult to achieve. Ventilation, cleaning, hand hygiene, and personal protective equipment are the most important protective measures to limit and control exposure to C. psittaci. Adequate information, communication, and health surveillance belong to the responsibilities of the occupational physician. Future challenges lay in the rigorous reporting of infections in both poultry and poultry workers and in the development of an avian and human vaccine.

Fluorine Is a Major Constituent of the Marine Sponge Halichondria moorei.

Fluorine constitutes about 10 percent of the dry weight of the marine sponge Halichondria moorei. The fluorine occurs as potassium fluorosilicate, which is a potent anti-inflammatory agent. A closely related sponge living in the same habitat does not contain any fluorine. The habitat was found to be free of fluorine except for the small amount naturally present in seawater.

Immunity to avian influenza A viruses.

While the basic principles of immunity to the influenza A viruses are probably similar for all vertebrates, detailed understanding is based largely on experiments in laboratory mice. Elements of the innate response limit early virus replication, although high pathogenicity strains can trigger effusive cytokine/chemokine production and lethal shock. Virus clearance is normally mediated via CD8+ effector T cells but, in their absence, the class-switched antibody response can ultimately achieve the same goal. Protection against reinfection is optimally provided by antibody (IgG and IgA) specific for the homologous viral haemagglutinin, and priming against the neuraminidase and the low abundance, conserved M2 protein can also have an effect. Influenza virus-specific plasma cells and CD8+ T cells persist in the long term and the recall of the CD8+ T cell response can lead to earlier virus clearance. The characteristics of the aging immune system and possible, novel vaccine strategies are also considered.

Temporal changes in hematologic markers after splenectomy, splenic embolization, and observation for trauma.

INTRODUCTION: The spleen is one of the most commonly injured abdominal solid organs during blunt trauma. Modern management of splenic trauma has evolved to include non-operative therapies, including observation and angioembolization to preclude splenectomy in most cases of blunt splenic injury. Despite the shift in management strategies, relatively little is known about the hematologic changes associated with these various modalities. The aim of this study was to determine if there are significant differences in hematologic characteristics over time based on the treatment modality employed following splenic trauma. We hypothesized that alterations seen in hematologic parameters would vary between observation (OBS), embolization (EMB), and splenectomy (SPL) in the setting of splenic injury. METHODS: An institutional review board-approved, retrospective study of routine hematologic indices examined data between March 2000 and December 2014 at three academic trauma centers. A convenience sample of patients with splenic trauma and admission lengths of stay >96 h was selected for inclusion, resulting in a representative sample of each sub-group (OBS, EMB, and SPL). Basic demographics and injury severity data (ISS) were abstracted. Platelet count, red blood cell (RBC) count and RBC indices, and white blood cell (WBC) count with differential were analyzed between the time of admission and a maximum of 1080 h (45 days) post-injury. Comparisons between OBS, EMB, and SPL groups were then performed using non-parametric statistical testing, with statistical significance set at p < 0.05. RESULTS: Data from 130 patients (40 SPL, 40 EMB, and 50 OBS) were analyzed. The median age was 40 years, with 67 % males. Median ISS was 21.5 (21 for SPL, 19 for EMB, and 22 for OBS, p = n/s) and median Glasgow Coma Scale (GCS) was 15. Median splenic injury grade varied by interventional modality (grade 4 for SPL, 3 for EMB, and 2 for OBS, p < 0.05). Inter-group comparisons demonstrated no significant differences in RBC counts. However, mean corpuscular volume (MCV) and RBC distribution width (RDW) were elevated in the SPL and EMB groups (p < 0.01). Similarly, EMB and SPL groups had higher platelet counts than the OBS group (p < 0.01). In aggregate, WBC counts were highest following SPL, followed by EMB and OBS (p < 0.01). Similar trends were noted in neutrophil and monocyte counts (p < 0.01), but not in lymphocyte counts (p = n/s). CONCLUSION: This study describes important trends and patterns among fundamental hematologic parameters following traumatic splenic injuries managed with SPL, EMB, or OBS. As expected, observed WBC counts were highest following SPL, then EMB, and finally OBS. No differences were noted in RBC count between the three groups, but RDW was significantly greater following SPL compared to EMB and OBS. We also found that MCV was highest following OBS, when compared to EMB or SPL. Finally, our data indicate that platelet counts are similarly elevated for both SPL and EMB, when compared to the OBS group. These results provide an important foundation for further research in this still relatively unexplored area.

MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates.

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T-cell receptor contact-residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both Bacillus Calmette-Guerin vaccination and Mycobacterium tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

The dissimilar interactions of the calcium antagonist flunarizine with different phospholipid classes and molecular species: a differential scanning calorimetry study.

The influence of the class IV calcium antagonist flunarizine on the phase behaviour of different species of the major phospholipid classes of mammalian plasma membranes has been examined using differential scanning calorimetry. We show that it has the ability to substantially influence the phase behaviour of phospholipids. Flunarizine significantly influences the gel to liquid-crystalline transition temperature of phosphatidylserines whilst having little effect on those of the phosphatidylethanolamines tested. The liquid-crystalline to inverted hexagonal phase transition of phosphatidylethanolamines is, however, strongly induced by the presence of flunarizine. Examination of the effect of flunarizine on the phase behaviour of different phosphatidylcholine species revealed an acyl-chain dependent influence. Dissimilar results with phosphatidylcholines, phosphatidylethanolamines and phosphatidylserines reveal different locations and ionization states for the drug in the different phospholipid bilayers. These results not only indicate an essential role for the ionization state of the drug in determining drug-phospholipid interactions but also the role of the phospholipid in determining the ionization state of the drug and have important implications for drug-membrane interactions demonstrating that drug interaction with one phospholipid may bear no relation whatsoever to its interaction with another.

Prevention of calcium-induced membrane structural alterations in erythrocyte membranes by flunarizine.

The calcium antagonist flunarizine is shown to be able to prevent particle aggregation, membrane aggregation and blebbing resulting from elevated calcium concentrations. The anti-ischemic effects of flunarizine may therefore result in part from its ability to directly interfere with calcium-membrane interactions and thus prevent the lethal membrane reorganizations which occur after a period of ischemia during intracellular calcium overload.

Electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering.

The dielectric constant in the active site cleft of subtilisin from Bacillus amyloliquefaciens has been probed by mutating charged residues on the rim and measuring the effect on the pKa value of the active site histidine (His64) by kinetics. Mutation of a negatively charged surface residue, which is 12 to 13 A from His64, to an uncharged one Asp----Ser99) lowers the pKa of the histidine by up to 0.4 unit at low ionic strength (0.005 to 0.01 M). This corresponds to an apparent dielectric constant of about 40 to 50 between Asp99 and His64. The mutation is in an external loop that is known to tolerate a serine at position 99 from homologies with subtilisins from other bacilli. The environment between His64 and Asp99 is predominantly protein. Another charged residue that is at a similar distance from His64 (14 to 15 A) and is also in an external loop that is known to tolerate a serine residue is Glu156, at the opposite side of the active site. There is only water in a direct line between His64 and Glu156. Mutation of Glu----Ser156 also lowers the pKa of His64 by up to 0.4 unit at low ionic strength. This change again corresponds to an apparent dielectric constant of about 40 to 50. The pKa values were determined from the pH dependence of kcat/KM for the hydrolysis of peptide substrates, with a precision of typically +/- 0.02 unit. The following suggests that the changes in pKa are real and not artefacts of experimental conditions: Hill plots of the data for pKa determination have gradients (h) of -1.00(+/- 0.02), showing that there are negligible systematic deviations from theoretical ionization curves involving a monobasic acid: the pH dependence for the hydrolysis of two different substrates (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl p-nitroanilide and benzoyl-L-valyl-L-glycyl-L-arginyl p-nitroanilide) gives identical results so that the pKa is independent of substrate; the pH dependence is unaffected by changing the concentration of enzyme, so that aggregation is not affecting the results; the shift in pKa is masked by high ionic strength, as expected qualitatively for ionic shielding of electrostatic interactions.

Isolation of a putative receptor from Zea mays microsomal membranes that interacts with the G-protein, GP alpha 1.

The C-terminal region of a heterotrimeric G-protein alpha-subunit is known to be one of the principal determinants governing its interaction with its cognate receptor. Use of an oligopeptide corresponding to the fifteen C-terminal residues of the Arabidopsis G alpha-subunit (GP alpha 1), as an affinity ligand, led to the resolution of a tightly binding 37 kDa membrane polypeptide from detergent solubilised Zea microsomal fraction membranes. An identical polypeptide bound tightly to an affinity matrix containing recombinant GP alpha 1 protein as ligand: binding and release of this 37 kDa protein was dependent on the activation state of GP alpha 1 which was regulated by inclusion or omission of the G-protein activator AlF-4. Finally, the isolated 37 kDa protein was labelled with the lectin concanavalin A, indicating it to be glycosylated. These data are consistent with the identity of the 37 kDa membrane polypeptide as a receptor that interacts with the Zea homologue of GP alpha 1.

A controlled study of amitriptyline in the treatment of chronic pain.

This paper reports a study in which a double-blind controlled cross-over study of amitriptyline vs. placebo was carried out in a group of patients referred to a multidisciplinary pain clinic for the management of chronic intractable pain for which no substantial organic cause could be demonstrated. Of 52 patients entering the 12-week trial, 20 withdrew before completion. No differences were found in terms of global improvement on either agent. Subjective reports indicated a greater reduction in pain at 2 and 4 weeks on amitriptyline, but no difference at 6 weeks. None of the baseline measures was predictive of response.

Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae in vitro.

Two bioassays were used to evaluate the interaction of fresh and cryopreserved equine semen with oviductal epithelial cells (OEC) and with the zona pellucida (ZP). Split ejaculates were either stored at room temperature or frozen and thawed. In experiment 1, progressive motility and membrane integrity were evaluated for each treatment. Fluorescent labeled spermatozoa were cocultured with monolayers of OEC for 30 minutes, and the number of sperm attached to OEC was counted by fluorescence microscopy and analysis of digitized images. Motility of spermatozoa attached to OEC was observed at 0.5, 3, 6, 18, 24, and 48 hours after insemination. In experiment 2, progressive motility, membrane integrity, and acrosomal integrity were determined. Differential labeling with the fluorochromes fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) was used to distinguish fresh and frozen-thawed spermatozoa. Equal numbers of motile spermatozoa from each treatment were incubated with salt-stored equine oocytes for 4 hours, and the number of spermatozoa firmly bound to the ZP was counted using dual-wavelength epifluorescence microscopy. Fewer (P < 0.001) cryopreserved spermatozoa attached to OEC compared to spermatozoa stored at room temperature. The motility of spermatozoa attached to OEC decreased over time within each treatment group (P < 0.001), but this decrease was not different between treatments. The mean number of spermatozoa bound per ZP and percentage of acrosome-intact spermatozoa were lower (P < 0.05) for frozen-thawed than for fresh spermatozoa. There was no effect of stallion on acrosomal status of frozen-thawed spermatozoa; however, the number of spermatozoa bound per ZP was different between stallions within treatments (P < 0.05). These results indicate that the ability of cryopreserved equine spermatozoa to attach to equine OEC or ZP in vitro is reduced compared to fresh extended spermatozoa due to changes other than a reduction in post-thaw motility or membrane integrity. The decreased ability of frozen-thawed spermatozoa to attach to OEC or to ZP could explain, in part, the reduced fertility of cryopreserved compared to fresh spermatozoa in the horse.

Mesothelioma in Great Britain in 1968-1983.

The British mesothelioma register records deaths in Great Britain when the word "mesothelioma" is on the death certificate. In 1968-1983 the mesothelioma deaths among men increased from 114 to 467, while those among women increased from 38 to 90. In 1983 the crude mesothelioma death rates were 17.5 per million and 3.2 per million for the men and women, respectively. The Northern region had the highest crude rates. At the county level, the highest crude deaths rates in 1976-1983 were recorded for the men in Devon and for the women in Lancashire. Marked differences occurred in the ratio of deaths among men to deaths among women for mesothelioma of the pleura (4.6:1) and for mesothelioma of the peritoneum (2:1). The age-specific death rates for men and women diverged markedly for pleural mesothelioma but not for peritoneal mesothelioma. Trends in the use of asbestos and in age- and sex-specific death rates suggest that the annual number of mesothelioma deaths will continue to increase, possibly until the turn of the century. This increase will be concentrated among the men as the main asbestos exposure of women occurred during the war and the annual deaths due to this exposure may have already peaked.

A mortality study of vinyl chloride monomer workers employed in the United Kingdom in 1940-1974.

The mortality experience of 5,498 male workers employed for at least one year during 1940-1974 in the vinyl chloride industry of the United Kingdom was followed through to 31 December 1984. There was a significant excess of nonsecondary liver tumors with 11 deaths, of which seven were angiosarcomas. All the angiosarcoma deaths occurred in autoclave workers with a median latency of 25 years from date of first exposure. A strong healthy worker effect was seen. Other than that for liver cancer, no increased incidence of cancer deaths attributable to vinyl chloride monomer exposure was found. There was no evidence of increased mortality from chronic liver disease. The incidence of death from respiratory disease was low and was not affected by polyvinyl chloride dust exposure.

Incidence and treatment of abnormal postpartum ovarian function in dairy cows.

The objectives of this study were to determine 1) the incidence of abnormal postpartum ovarian function in a large dairy herd in North Central Florida and 2) the effectiveness of gonadotrophin releasing hormone (GnRH) in treating this condition. The study was conducted from April 1988 to June 1989. The internal genitalia of the cows were initially examined per rectum (Day 0) between 19 and 29 (23 +/- 0.25) d after calving and again 14 d later (Day 14) for evidence of uterine involution and ovarian activity. The presence of a palpable corpus luteum (CL) and retrospective determination of plasma progesterone (P4) concentrations > 1 ng/ml were the criteria used to assess ovarian activity. Cows possessing a palpable CL and P4 concentrations > 1 ng/ml on Day 0 were determined to be cycling normally. A total of 1356 cows was used in this study. On Day 0, two groups were formed: Group 1 consisted of normal, cyclic cows, Group 2 of noncyclic cows. On Day 0, alternate cows in Group 2 were treated with GnRH (100 microg i.m). On Day 14, the previously nontreated cows in Group 2 were further divided into two groups, forming Group 3, nontreated cows and Group 4, cows treated with GnRH at this time. Group 5 was comprised of cows from Group 2 that did not respond to treatment with GnRH on Day 0; these cows were treated on Day 14 with GnRH (100 microg i.m). Group 6 was comprised of nontreated cows from Group 2 that responded spontaneously (presence of a CL) by Day 14. Reproductive parameters evaluated were the percentage of cows pregnant within 180 d after calving and at the end of the study, the number of days open and the number of services per conception. Data were statistically analyzed using Chi square and survival analysis. The results of this study indicate that the incidence of abnormal postpartum ovarian function in this herd was 30.2% and that the nontreated cows experienced more days open and required more services per conception than the treated cows, those that were cycling normally on the initial examination, and those that responded spontaneously by Day 14.

Role of carbohydrates in the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells in vitro.

OBJECTIVE: To test the hypotheses that the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells (OEC) in vitro is mediated by glycoproteins, and that proteins with carbohydrate-binding properties are present in the periacrosomal plasma membrane of equine spermatozoa. ANIMALS: 4 reproductively sound stallions, and 1 mare in estrus. PROCEDURES: In experiment 1a, fluorescent-labeled spermatozoa were cocultured with monolayers of OEC in the presence of 50 mM glucose, fructose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid, or 10 mg of fetuin or asialofetuin/ml in modified Tyrode's solution (TALP), or in TALP alone. After 2 hours of coculture, numbers of attached spermatozoa were counted by fluorescence microscopy and analysis of digitized images. In experiment 1b, progressive motility, viability, acrosomal integrity, and capacitation status were determined in spermatozoa incubated for 2 hours in the presence of the respective monosaccharides and glycoproteins or in TALP alone. In experiment 2, proteins isolated from the periacrosomal plasma membrane of equine spermatozoa were subjected to galactose affinity chromatography and subsequent one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. RESULTS: Numbers of spermatozoa attached to OEC were reduced (P < 0.05) after all treatments except N-acetyl glucosamine, compared with incubation in TALP alone. The lowest numbers of spermatozoa were bound in cultures incubated in the presence of galactose and asialofetuin. Spermatozoal motility was lower (P < 0.05) after incubation for 2 hours in the presence of fetuin, compared with control, and incubation in the presence of fetuin or asialofetuin caused a significant (P < 0.05) increase in the percentage of capacitated spermatozoa, compared with control. Affinity chromatography of periacrosomal plasma membrane proteins revealed a galactose-binding protein of about 66 kd. CONCLUSION: Recognition of glycoconjugates with exposed galactosyl residues on OEC by galactose-binding proteins on the periacrosomal plasma membrane of equine spermatozoa could mediate the attachment of equine spermatozoa to OEC in vitro.