Milk osteopontin retains integrin-binding activity after in vitro gastrointestinal transit.

Osteopontin (OPN) is a multifunctional protein highly expressed in milk, where it is hypothesized to be involved in immunological signaling via the conserved Arg-Gly-Asp (RGD) integrin-binding sequence. Intervention studies have indicated beneficial effects of orally administered OPN in animal and human infants, but the mechanisms underlying these effects are not well described. To induce physiological effects, OPN must resist gastrointestinal transit in a bioactive form. In this study, we subjected bovine milk OPN to in vitro gastrointestinal transit, and characterized the generated fragments using monoclonal antibody and mass spectrometric analyses. We found that the fragment Trp27-Phe151 containing the integrin-binding RGD sequence resisted in vitro gastric digestion. This resistance was dependent on glycosylation of threonine residues near the integrin-binding sequence in both human and bovine milk OPN. Furthermore, the fragment Trp27-Phe151 retained the ability to interact with integrins in an RGD-dependent process. These results suggest a mechanism for how ingested milk OPN can induce physiological effects via integrin signaling in the intestine.

Structure, function and control of complement C5 and its proteolytic fragments.

As part of the innate immune system, the complement system recognises a wide range of non-self structures present on pathogens or altered self cells. Its activation elicits proteolytic cascades which eventually results in the cleavage of the C5 protein into two fragments, C5a and C5b. The small anaphylatoxin C5a induces a variety of biological responses upon binding to the 7TM receptors C5aR and the C5L2, while the large C5b fragment nucleates formation of the membrane attack complex capable of killing susceptible pathogens by the formation of a pore structure in association with complement components C6, C7, C8, and C9. A number of regulatory molecules help to control C5 mediated immune responses towards host cells, but in several major inflammatory conditions including sepsis and arthritis, C5a is believed to contribute significantly to disease etiology. Inhibition of membrane attack complex assembly is already approved for treatment of paroxysmal nocturnal haemoglobinuria and atypical hemolytic uremic syndrome. A number of recent crystal structures have provided a comprehensive insight into the architecture and properties of intact C5 and its fragments, and how pathogens interfere with their function. Here we review the functional and structural aspects of C5 and its fragments, the pathological conditions associated with them, and strategies employed by pathogens to interfere with the biological function of C5. Structural insight and elucidation of evasion strategies employed by pathogens present a unique opportunity for promoting the development of novel selective C5 inhibitors with therapeutic applications.

An assay for the mannan-binding lectin pathway of complement activation.

The mannan-binding lectin (MBL) pathway of complement activation has been established as the third pathway of complement activation. MBL is a carbohydrate-binding serum protein, which circulates in complex with serine proteases known as mannan-binding lectin associated serine proteases (MASPs). When bound to microorganisms, the MBL complex activates the complement components C4 and C2, thereby generating the C3 convertase and leading to opsonisation by the deposition of C4b and C3b fragments. This C4/C2 cleaving activity is shared with the C1 complex of the classical pathway of complement activation. Therefore, in a generally applicable complement activation assay specific for the MBL pathway, the activity of the classical pathway must be inhibited. This can be accomplished by exploiting the finding that high ionic strength buffers inhibit the binding of C1q to immune complexes and disrupt the C1 complex, whereas the carbohydrate-binding activity of MBL and the integrity of the MBL complex is maintained under hypertonic conditions. In the assay described here, the specific C4b-depositing capacity of the MBL pathway was determined by incubating serum diluted in buffer containing 1 M NaCl in mannan-coated microtiter wells before the addition of purified C4. The interassay coefficient of variation in the ELISA version was 7.3%. As expected no activity was found in MBL-deficient serum. When 100 normal serum samples were analysed we found that the MBL level correlated with the amount of C4b deposited on the mannan-coated surface. However, we also found a threefold variation in C4b-depositing capacity between individuals with similar MBL concentrations. The assay permits for the determination of MBL complex activity in serum and plasma samples and may thus be used to evaluate the clinical implications of complement activation via this pathway.

The mannan-binding lectin pathway of complement activation: biology and disease association.

Mannan-binding lectin (MBL) is a plasma protein found in association with several serine proteases (MASPs) forming the MBL complex. MBL recognises carbohydrate structures arranged in a particular geometry, such as those found on the surface of micro-organisms. When bound to e.g. bacteria the MBL complex will initiate the activation of the complement cascade. Mounting evidence supports the importance of the MBL pathway of complement activation in innate immunity. In this review, we focus on the structure and function of the proteins within the MBL pathway and address the properties of the pathway as an initiator of the host response against potential pathogenic micro-organisms.

Characterization and quantification of mouse mannan-binding lectins (MBL-A and MBL-C) and study of acute phase responses.

Rat monoclonal antibodies (MoAbs) against mouse mannan-binding lectin (MBL)-A and MBL-C were generated and assays for MBL-A and MBL-C were constructed. This allowed for the quantitative analysis of both proteins for the first time. Previously only MBL-A has been quantified using less standardized methods. In a mouse serum pool the concentrations were now determined at 7.5 microg MBL-A and 45 microg MBL-C per ml. On gel permeation chromatography of mouse serum, MBL-A eluted corresponding to a M(r) of 850 kDa whereas the majority of MBL-C eluted corresponding to a Mr of 950 kDa. On sucrose density gradient centrifugation the sedimentation velocities of MBL-A and MBL-C were estimated at 7.3 S and 10.8 S, respectively. The MBL-A and MBL-C levels in 10 laboratory mice strains were compared and found to vary between 4 microg/ml to 12 microg/ml, and 16 microg/ml to 118 microg/ml, respectively. After the induction of acute phase responses by intraperitoneal injection of either casein or lipopolysaccharide (LPS), MBL-A was found to increase approximately two-fold, with a maximum after 32 h, while MBL-C did not increase significantly. In comparison, serum amyloid A component (SAA) peaked at 15 h with an approximate 100-fold increase.

Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2.

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.

Interaction of C1q and mannan-binding lectin (MBL) with C1r, C1s, MBL-associated serine proteases 1 and 2, and the MBL-associated protein MAp19.

Mannan-binding lectin (MBL) and C1q activate the complement cascade via attached serine proteases. The proteases C1r and C1s were initially discovered in a complex with C1q, whereas the MBL-associated serine proteases 1 and 2 (MASP-1 and -2) were discovered in a complex with MBL. There is controversy as to whether MBL can utilize C1r and C1s or, inversely, whether C1q can utilize MASP-1 and 2. Serum deficient in C1r produced no complement activation in IgG-coated microwells, whereas activation was seen in mannan-coated microwells. In serum, C1r and C1s were found to be associated only with C1q, whereas MASP-1, MASP-2, and a third protein, MAp19 (19-kDa MBL-associated protein), were found to be associated only with MBL. The bulk of MASP-1 and MAp19 was found in association with each other and was not bound to MBL or MASP-2. The interactions of MASP-1, MASP-2, and MAp19 with MBL differ from those of C1r and C1s with C1q in that both high salt concentrations and calcium chelation (EDTA) are required to fully dissociate the MASPs or MAp19 from MBL. In the presence of calcium, most of the MASP-1, MASP-2, and MAp19 emerged on gel-permeation chromatography as large complexes that were not associated with MBL, whereas in the presence of EDTA most of these components formed smaller complexes. Over 95% of the total MASPs and MAp19 found in serum are not complexed with MBL.

Control of the classical and the MBL pathway of complement activation.

The activation of complement via the mannan-binding lectin (MBL) pathway is initiated by the MBL complex consisting of the carbohydrate binding molecule, MBL, two associated serine proteases, MASP-1 and MASP-2, and a third protein, MAp19. In the present report we used an assay of complement activation specifically reflecting the physiological activity of the MBL complex to identify biological and synthetic inhibitors. Inhibitor activity towards the MBL complex was compared to the inhibition of the classical pathway C1 complex and to a complex of MBL and recombinant MASP-2. A number of synthetic inhibitors were found to differ in their activities towards complement activation via the MBL pathway and the classical pathway. C1 inhibitor inhibited both pathways whereas alpha2-macroglobulin (alpha2M) inhibited neither. C1 inhibitor and alpha2M were found to be associated with the MBL complex. Upon incubation at 37 degrees C in physiological buffer, the associated inhibitors as well as MASP-1, MASP-2, and MAp19 dissociated from MBL, whereas only little dissociation of the complex occurred in buffer with high ionic strength (1 M NaCl). The difference in sensitivity to various inhibitors and the influence of high ionic strength on the complexes indicate that the activation and control of the MBL pathway differ from that of the classical pathway. MBL deficiency is linked to various clinical manifestations such as recurrent infections, severe diarrhoea, and recurrent miscarriage. On the other hand, impaired control of complement activation may lead to severe and often chronically disabling diseases. The results in the present report suggests the possibility of specifically inhibiting of the MBL pathway of complement activation.

[Surgical treatment of snoring and sleep apnea]. / Kirurgisk behandling af snorken og søvnapnø.

Twenty-one patients (four females and 17 males) greatly inconvenienced by snoring were subjected to UPP with CO2-laser technique. All patients were overweight (body-mass index average 30 kg/m2). Narrowing was found at the naso/oropharyngeal border in 71%. All had either abnormal uvula or soft palate. Snoring was reduced to a degree no longer socially troublesome in 17 patients. Three patients were still inconvenienced to some extent. Snoring was completely unchanged in only one patient. UPPP is not very difficult to perform, and we did not notice any sequelae of importance. We think that UPPP is an effective operation for severe snoring, although it does not eliminate the symptom.

[CO2 laser treatment of proximal pharyngo-esophageal diverticula (Zenker's diverticula)]. / CO2-laser behandling af proksimale faryngo-øsofageale divertikler (Zenker's divertikel).

The pharyngo-esophageal diverticulum (Zenker's diverticulum) has traditionally been treated operatively by an external approach. Since the 1950's the endoscopic technique has been performed increasingly as a good alternative. Since 1989, we have treated Zenker's diverticulum endoscopically as a standard procedure, and hereby we present our results here. We use the Benjamin-Hollinger diverticuloscope, through which the septum between the diverticulum and esophagus is divided by a CO2-laser. Nine patients have been treated, five men and four women. Mean age 77 years (range 59-93). Diverticulum size varied between 1x2 cm and 6x6.5 cm. No major complications occurred. One patient had recurrence of symptoms after three months and was reoperated with the same technique. The mean observation period is 12 months (range 4-28). We find the endoscopic CO2-laser technique safe, and for the elderly age group suffering from Zenker's diverticulum, less traumatic than the external approach.

Results of uvulopalatopharyngoplasty in snoring.

Twenty-one patients (4 females and 17 males) with very annoying snoring were subjected to UPPP with CO2-laser technique. All patients were overweight (bodymass index average 30.3 kg/m2). In 71%, narrowing were found at the naso/oropharyngeal border. All had either abnormal uvula or soft palate. In 17 patients, snoring was reduced to a degree no longer socially annoying. Three were annoying to some extent. In only one patient, snoring was completely unchanged. UPPP is not very difficult to perform and we did not notice any troublesome sequelae. We think that UPPP is an effective operation for severe snoring, although it does not eliminate the symptom.

[Functional endoscopic endonasal sinus surgery. First experiences with a new method]. / Funktionel endoskopisk endonasal bihulekirurgi. De første erfaringer med en ny metode.

A material of 46 patients who were treated operatively on account of chronic recurrent sinusitis by functional endoscopic sinus surgery by Messerklinger's method is presented. In 80% the operation was bilateral. As a serious complication, one case of dural lesion occurred. This healed without further complications and without sequelae. Just under 30% experienced slight per- or postoperative complications in the form of haemorrhage and infection. All of the patients participated in a follow-up examination after an average of 13 months' observation. 80-85% stated that they were free from symptoms or had improved after operation. Late complications were insignificant. Half of the patients had synechia formation in the resection area but the presence of synechia was not found to have any significant relationship to the postoperative course. When it is taken into consideration that the first experience with a new method is concerned, the results are considered to be satisfactory. A more extensive patient material with more prolonged observation and experience from other departments are necessary before the employability of the method in the Danish therapeutic system can be assessed.

[Thyroid surgery performed at a head and neck oncologic department]. / Thyreoideakirurgi på en hoved-hals-onkologisk afdeling.

During a period of ten years, 1977-1986, 778 patients were treated for diseases of the thyroid gland. Of these, 678 were benign and 100 malignant. The reasons for referral were most frequently a lump in the thyroid gland, possibly with suspected malignant changes. In the group with benign conditions, there were five times as many women as men whereas the proportion in the malignant group was 1.5 to 1. Technetium-scintigraphy and fine-needle aspiration were undertaken routinely preoperatively. As a rule, none of the preoperative investigations could reveal with certainty whether the condition was benign or malignant but, in certain cases, the investigations may provide indication and guidance for the continued treatment or control of the patients concerned. Treatment with particular attention to the recurrent nerve and the complications, particularly recurrent paresis, is mentioned.

Fine-needle aspiration biopsy of the thyroid gland.

Fine-needle aspiration biopsy is a diagnostic procedure used routinely in the evaluation of patients referred with a thyroid swelling. The technique and the results in our series of 233 patients, which included 39 carcinomas, are presented. A relatively high diagnostic specificity of 81% was found, whereas the diagnostic sensitivity was considerably lower (58%). It is concluded that fine-needle aspiration biopsy is a valuable diagnostic procedure in the preoperative assessment of patients with thyroid swellings. A definitive treatment, however, must never be based entirely on the results of a fine-needle aspiration biopsy. It is concluded that the results can be improved if the procedure is performed by a few highly skilled clinicians and pathologists.

Thyroid surgery.

In a 5-year period we treated 233 patients referred to us with a thyroid swelling. Among them, 39 patients (17%) had a malignant tumor. Seventy-eight percent of the patients were women. The preoperative examination procedure is discussed. Preoperatively, recurrent nerve palsy was found in 2.6% of patients with benign tumors and in 28% of patients with malignant tumors. The histologic features of the benign and the malignant tumors are described. The operative procedure in the benign group consisted of enucleation, resection, or lobectomy. In the patients with malignant tumors a radical operation was performed, consisting of lobectomy (unilateral or bilateral) when possible, eventually combined with radical neck dissection. The operative complications are discussed.