Brief report: first identification of H4 histamine receptor in healthy salivary glands and in focal sialadenitis in Sjögren's syndrome.

OBJECTIVE: The conventional H(1) and H(2) histamine receptors have >10,000-fold lower avidity for histamine than H(4) histamine receptor, which has been implicated in autoimmune diseases. This study was undertaken to compare H(4) histamine receptor levels in the salivary glands (SGs) of healthy controls with those in the SGs of patients with primary Sjögren's syndrome (SS). METHODS: H(4) histamine receptor messenger RNA (mRNA) was analyzed using real-time quantitative polymerase chain reaction, and the receptor protein was examined using immunostaining. Effects of the H(4) histamine receptor agonist ST-1006 on cytokine synthesis by human SG (HSG) cells were analyzed using xMAP technology and enzyme-linked immunosorbent assay. RESULTS: Healthy SGs contained H(4) histamine receptor mRNA. The receptor protein was localized to the acinar and ductal epithelial cells. H(4) histamine receptor agonist stimulated HSG cells to produce the cytokines interleukin-8 and vascular endothelial growth factor. SS patients had low H(4) histamine receptor levels. CONCLUSION: H(1) and H(2) histamine receptor antagonists are not effective in the treatment of autoimmune diseases. However, such antagonists do not affect the newly discovered H(4) histamine receptor. Dendritic cells and lymphocytes are nonprofessional histamine-producing cells, which produce histamine at 100-1,000-fold lower rates than mast cells do. Saliva contains only 0.31-12.4 ng/ml histamine, which is too low to stimulate H(1) or H(2) histamine receptor, but stimulates H(4) histamine receptor half maximally. Our findings show that H(4) histamine receptor is strongly expressed in tubuloacinar SG cells, which emphasizes the role of these cells in the pathogenesis of SS.

Salivary glands - "an unisex organ'?

Usually no distinction is made between female and male salivary glands although cyclic changes of and / or differences in serum and salivary sex steroid concentrations characterize women and men. Moreover, sexual dimorphism is well recognized in salivary glands of rodents.Salivary glands contain estrogen and androgen receptors and are, according to modern high throughput technologies,subjected to gender differences not explainable by gene dose effects by the X chromosome alone. Because sex steroids are lipophilic, it is often thought that approximately 10% of them passively diffuse from plasma to saliva. Indeed, saliva can find use as sample material in sports medicine, pediatrics, veterinary medicine and behavioral sciences. Last but not least, humans and other primates are unique in that they have a reticular zone in their adrenal cortex, which produces dehydroepiandrosterone and androstendione pro-hormones. These are processed in peripheral tissues, not only in female breast and uterus and male prostate, but also in salivary glands by an intracrine enzymatic machinery to active 17b-estradiol,dihydrotestosterone and others, to satisfy and buffer against a constantly changing needs caused by circadian,menstrual, pregnancy and chronobiological hormonal changes in the systemic circulation. Female dominance of Sjögren's syndrome and certain forms of salivary gland cancer probably reflect these gender-based differences.

Receptor activator of nuclear factor kappa B ligand in an experimental intervertebral disc degeneration.

OBJECTIVE: This study was designed to clarify the role of the receptor activator of nuclear factor kappa B ligand (RANKL) in the process of discus degeneration and spondylarthrosis. It was hypothesized that experimental discus lesion would initiate not only local bone remodelling but also increased osteoclast formation on a location remote to the injury site due to altered spinal biomechanics. It was speculated that these changes in vertebral bone remodelling could be reflected in an increased RANKL expression. METHODS: The presence of RANKL in the spine was studied in an experimental perforating lesion of the cranial endplate of L4 and the adjoining disc in six domestic pigs and in one human herniated disc. After three months, the experimental and contiguous control vertebrae, complete with intervertebral discs, were subjected for immunohistochemistry. RESULTS: This is the first study to show that RANKL was locally seen (produced) in osteoblasts, fibroblasts replacing annulocytes and mesenchymal bone marrow cells and, in part, apparently bound to the surface of osteoclasts and macrophage-like prefusion macrophages. Such RANKL induction was also seen at sites remote from the experimental lesion driving the whole process. More RANKL-positive cells were found in close proximity to the endplate than in the central parts of the vertebrae. Osteocytes in bone matrix and most bone marrow cells in the marrow microenvironment showed no RANKL staining. Human annulus fibrosus also contained RANKL, RANK and OPG. CONCLUSIONS: We have demonstrated that RANKL is produced locally, also in soluble form, at the site of injury and also in intact vertebrae and bony structures likely due to altered biomechanics. It seems to be engaged in bone healing and remodelling, essentially proving our working hypothesis. These secondary bone changes could represent part of the degenerative spine disease (spondylarthrosis). RANKL inhibitors, like recombinant human osteoprotegerin (OPG), could be interesting drugs to test, not only in osteoporosis, but also in spondylarthrosis.

Pro-inflammatory, pleiotropic, and anti-inflammatory TNF-alpha, IL-6, and IL-10 in experimental porcine intervertebral disk degeneration.

The aim of this study was to check the balance between tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-10 (IL-10) in well-developed end-stage disk disease in the disk itself as well as in paradiskal spine. In 6 domestic pigs the cranial bony end plate of the L4 vertebra was perforated to the nucleus pulposus. At 3 months the degenerated experimental and contiguous control disks, together with the adjoining bony and cartilaginous vertebral end plates, bone marrow, and spinal ligaments, were excised and used for immunohistochemical analysis. In general, there were more TNF-alpha and in particular IL-10 positive cells in the degenerated disks than in the control disks, whereas the number of IL-6 labeled cells did not differ among sites or between control and experimental intervertebral disks. These results suggest that TNF-alpha and IL-10 are involved in the late reparatory phases of the experimental disk lesion. Use of an experimental model showed that strictly disk-directed manipulation and degeneration are also reflected in the contiguous vertebrae, including adjoining cartilage, bone, marrow, and ligaments.

Expression of vascular endothelial growth factor receptors coincide with blood vessel in-growth and reactive bone remodelling in experimental intervertebral disc degeneration.

OBJECTIVE: To analyze immunohistochemically the localization of the VEGF receptors in experimental intervertebral disc degeneration tissues in a pig model. MATERIALS AND METHODS: In six domestic pigs, the cranial bony endplate of the L4 vertebra were perforated into the nucleus pulposus. Three months postoperatively, the animals were sacrificed and the experimental and control vertebrae, complete with intervertebral discs, were excised and subjected for immunohistochemical staining of vascular endothelial growth factor receptors (VEGFR) along with VEGF - A, -C, -D and blood and lymphatic vessel markers vWF and LYVE-1. RESULTS: The results of immunohistochemical analysis of experimental samples showed VEGFR-1 (Flt-1) expression in intervertebral disc and all paradiscal tissues studied. In control samples expression of VEGFR-1 was lower and absent in the intervertebral discs. Comparatively less of VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4) than VEGFR-1was found in degenerated intervertebral discs and paradiscal tissues. In contiguous control intervertebral discs and control paradiscal tissues VEGFR-2 and-3 receptors were expressed to a lower extent than in experimental tissues or were even totally absent. Also growth factors VEGF-A, -C, -D, as well as von Willebrand factor and to a much lower extent LYVE-1 were differently expressed in experimental and control intervertebral discs. CONCLUSION: In experimental intervertebral disc degeneration, VEGF receptors were expressed in the damaged disc and paradiscal tissues. In the same tissues, VEGF-A, -C, and -D, signs of blood and lymphatic vessel in-growth and reactive/adaptive vertebral bone remodelling were found.

Low molecular weight heparin versus acenocoumarol in the secondary prophylaxis of deep vein thrombosis.

The aim of this study was to determine the efficacy and safety of subcutaneous weight-adjusted dose low molecular weight heparin (LMWH) compared with oral anticoagulant (OA) in the prevention of recurrent venous thromboembolism. In a prospective multicenter trial, 202 patients with symptomatic proximal deep vein thrombosis (DVT) were included. As soon as the diagnosis of DVT was confirmed by phlebography, 101 were randomly assigned to receive LMWH (nadroparin) for secondary prophylaxis and 101 to receive OA (acenocoumarol). Patients in both groups were initially treated with nadroparin in a dose of 85 anti-Xa IU/kg s.c. every 12 h. Secondary prophylaxis with either nadroparin, 85 anti-Xa IU/kg s. c. once daily, or acenocoumarol was continued for at least 3 months. Three patients in the LMWH group and 6 in the OA group were excluded from analysis for various reasons. During the one-year combined secondary prophylaxis and surveillance period, 7 of of the 98 evaluable patients (7.1%) in the LMWH group and 9 of the 95 evaluable patients (9.5%) in the OA group had a documented recurrence of venous thromboembolism (Fisher's exact test, p = 0.61). Of these, 2 patients who received LMWH and 7 patients on acenocoumarol had recurrences in the 3-month period of secondary prophylaxis. Four patients (4.1%) in the LMWH group developed bleeding complications during this study period, as compared with 7 (7.4%) in the OA group (Fisher's exact test, p = 0.37). There were two major bleedings, one in the LMWH group and one in the OA group. Eleven patients died, 5 (5.1%) in the LMWH group and 6 (6.3%) in the OA group. It is concluded that nadroparin in a dose of 85 anti-Xa IU/kg s.c. once daily provides an effective and safe alternative to oral anticoagulants in the secondary prophylaxis of DVT.

Studies on binding of HIV-1 p24gag peptide to HLA-Cw3+ cells.

Human major histocompatibility complex class I antigens, HLA-C, are expressed on the cell surface at approximately a tenfold lower level than HLA-A and -B. We hypothesized that the expression of HLA-C is limited by the quantity of high affinity peptides which bind to these molecules, thus allowing only a small fraction of HLA-C molecules to be transported and/or to remain stable on the cell surface. If this assumption is correct, then the addition of exogenous peptide should increase cell surface HLA-C expression. To verify the hypothesis, we pulsed lymphoblastoid cell line PAJ (HLA-Cw3+) with synthetic HIV-1 p24gag 145-152 peptide, known to be presented to T-lymphocytes by HLA-Cw3 molecule. PAJ (HLA-Cw3+) cells bound approximately two times more of the peptide than HAJ (HLA-Cw3-), and four times more than 500/C9 (HLA-Cw3-) cells. Accordingly, overnight pulsing of PAJ cells with the p24gag 145-152 peptide caused an increase in class I HLA expression detected on the cell surface by flow cytofluorimetric analysis with anti-HLA-B,C monoclonal antibodies but not by anti-HLA-A antibody. In contrast, HLA-Cw3- cells treated in the same manner did not show any increase of HLA class I expression. Our data suggest that low concentration of high affinity peptides within the cell may be one of the factors limiting cell surface expression of HLA-C molecules.

Increased but imbalanced expression of VEGF and its receptors has no positive effect on angiogenesis in systemic sclerosis skin.

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) and its vascular and lymphatic receptors in skin in systemic sclerosis (SSc) compared to systemic lupus erythematosus (SLE), Raynaud's phenomenon (RP) and normal healthy control skin. METHODS: Staining was performed using rabbit anti-human antibodies in DAKO TechMate Horizon staining robot programmed for the biotin-streptavidin protocol. RESULTS: VEGF was sporadically and weakly expressed in normal skin, but in spite of vascular damage in diseased skin, VEGF expression was only slightly upregulated. In contrast, its vascular receptors VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1), were clearly upregulated. Finally, the lymphatic VEGFR-3 (Flt-4) receptor was also upregulated in diseased skin and ectopically expressed also in blood vessels. Negative staining and positive sample controls confirmed the specificity of the staining. CONCLUSION: The imbalanced expression of VEGF and its vascular receptors suggest that the compensatory efforts to angiogenesis fail in SSc, in part due to insufficient local production of VEGF, which was low compared to VEGFR expression. This is compatible with the recent observations on the lack of alpha V beta 3+ newly formed blood vessels in SSc skin. Since microvascular angiogenic stimuli normally induce first VEGF and then VEGFR, these findings also suggest that the angiogenic cascade is turned on, but there is a defect in the finalization of its effects. Normalization of angiogenic cascade in SSc could provide a future therapeutic target.

Dual effects of caspase-1, interleukin-1 beta, tumour necrosis factor-alpha and nerve growth factor receptor in inflammatory myopathies.

OBJECTIVE: To analyse the expression of factors potentially involved in skeletal muscle degeneration and regeneration in dermatomyositis (DM), systemic sclerosis (SSc), polymyositis (PM), systemic lupus erythematosus (SLE) and non-inflammatory myopathies. METHODS: Immunohistochemical staining of skeletal muscle biopsies (10 DM, 10 SSc, 10 PM, 10 SLE, 10 non-inflammatory myopathies) for tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), activated caspase-1, pan-macrophage marker CD68, inducible nitric oxide synthase (NOS2) and nerve growth factor receptor (NGFR). TechMate staining robot and biotin-streptavidin protocol were used. RESULTS: Expression of TNF-alpha, IL-1 beta, caspase-1 and NOS2 was found in the cytoplasm and sarcolemma of dystrophic skeletal muscle fibres. TNF-alpha and IL-1 beta immunoreactive profiles were faint and few and close to satellite nuclei-containing regenerating muscle fibres both in inflammatory and non-inflammatory myopathies. NGFR expression was found in comparable areas. In non-inflammatory inherited myopathies more nuclei were caspase-1 immunoreactive whereas caspase-1 expression was rarely seen in inflammatory myopathies, implying regeneration of the affected muscle fibres. CONCLUSION: Prominent expression of the proinflammatory factors TNF-alpha, IL-1 beta and NOS2 and caspase-1 is associated with muscle fibre damage, albeit when expressed to a low degree these factors may, like NGFR, contribute to muscle regeneration and healing.

Rabbit antibodies to some sequences of human chorionic gonadotropin and their use for immunoassay of the hormone.

Three synthetic peptides corresponding to amino acid sequences of two human chorionic gonadotropin (hCG) subunits were conjugated to thyroglobulin and used for immunization of rabbits. The highest antibody concentration was found in antisera from rabbits immunized with the peptide corresponding to 122-145 sequence of the hormone beta-subunit. Specificity of antisera and immunoaffinity purified rabbit antibodies were studied. Antibody anti-122-145-beta hCG with antibody anti-beta hCG conjugated to peroxidase were suitable for sensitive and specific enzyme immunoassay of hCG and beta hCG. Good correlation was noted between assay of the hormone in sera of patients with trophoblastic diseases or in urine of pregnant women by ELISA with the rabbit antibody pair and by commercial kit or by ELISA with two monoclonal antibodies.

Goat antibodies to amino acid sequences of human chorionic gonadotropin (hCG)

Human chorionic gonadotropin, its two subunits and its conjugate with thyroglobulin were used for immunization. The strongest immunogens were demonstrated to be the intact hormone and beta subunit, the weakest was alpha subunit. Using three peptides corresponding to hormone subunits coupled to Sepharose 4B various antibodies were isolated from goat antiserum by immunoaffinity chromatography. Specificity of the purified antibody preparations was studied. It was demonstrated that enzyme linked immunosorbent assay with two goat antibodies--one for coating of microtiter plates and the other one conjugated with peroxidase--is suitable for sensitive assays of both human chorionic gonadotropin and luteinizing hormone. Specific and sensitive assays were performed using combination of goat antibody anti-122-145-beta hCG and monoclonal antibody anti-alpha hCG.

Search for antibodies for immunoenzymatic assay of human lutropin.

Five peptides corresponding to human lutropin (hLH) subunit fragments were synthesized by a solid phase method and their physicochemical characteristics are presented. Antibodies induced in rabbits with 3 peptides of beta hLH fragments coupled to thyrotropin did not bind hLH and human chorionic gonadotropin (hCG). Also 2 synthetic peptides of alpha hLH fragments did not react with rabbit anti-hLH, anti-hCG and anti-alpha hCG antibodies. Using 2 monoclonal anti-alpha hCG and anti-beta hLH antibodies a sensitive sandwich ELISA technique was elaborated. Using this technique stability of hLH was investigated. The ELISA was also applied for assays of urine hLH in normal and ovulation days.

The influence of D-Ala1-peptide T amide, an analogue of HIV glycoprotein 120 fragment, on the CD4--anti CD4 lymphocyte interaction.

D-Ala1-peptide T amide (DAPTA), synthetic analogue of the HIV glycoprotein 120 sequence, was used to study its binding to specific cellular receptor of HIV, CD4. The analogue contains eight aminoacid residues including 4 threonine residues; its molecular weight being 992. We have shown the temperature- and dose-dependent inhibitory effect of peptide T on the CD4 - anti CD4 binding. The strongest effect was noted at 37 degrees C with the peptide dose of 150 nM: 62% inhibition of binding. Other means of possible blocking of HIV attachment to the host cellular receptor are discussed.

Specific interactions of Cu2+ ions with fragments of envelope protein of hepatitis B virus.

Potentionmetric and spectroscopic (EPR, CD and absorption spectra) data have shown that a fragment of envelope proteins of the hepatitis B virus could be very specific bind molecules for Cu2+ ions using arginine lateral NH2 donor sites. The presence of Pro and Asp residues makes Arg binding not only very specific, but also very efficient.

Complexes of Cu(II) with Asn-Ser-Phe-Arg-Tyr-NH2; an example of metal ion-promoted conformational organization which results in exceptionally high complex stability.

The pentapeptide fragment of ANF, Asn-Ser-Phe-Arg-Tyr-NH2, coordinates to Cu(II) using the same four nitrogen donor centers as simple pentapeptides such as pentaalanine yet the complexes are of much higher stability as a result of a highly organized side-chain structure which is present in the complex but absent from the free ligand.

Vascular damage and lack of angiogenesis in systemic sclerosis skin.

The aim of this study was to analyse microvascular damage and compensatory angiogenesis in skin from patients with systemic sclerosis (SSc) compared with systemic lupus erythematosus (SLE), Raynaud's phenomenon (RP) and healthy controls. Immunohistochemistry was used for skin biopsies (9 SSc, 10 SLE, 9 RP and 12 healthy controls) using von Willebrand factor and beta3 integrin subunit specific antibodies, TechMate immunostaining robot and biotin-streptavidin protocol. In the early stages of SSc, vWF was found in the perivascular space and interstitial matrix in papillary but not in the reticular dermis, in particular around small oedematous blood vessels infiltrated by mononuclear cells. The extravascular release of vWF in SSc specimens was associated with weak or even a total lack of immunoreactivity within the associated endothelial cells. Late stages of SSc were characterised by loss of the dermal papillae, subepidermal fibrosis, hypovascularity and strong endothelial vWF expression without extravascular leakage. In all SSc patients studied only a few vascular profiles were weakly immunostained for beta3 integrin subunit. This work demonstrates that vWF is not only released into the systemic circulation, but is also leaked to the perivascular space/matrix. This local release and deposition of vWF is probably a sensitive and early marker of microvascular involvement in SSc pathogenesis. Local vWF release may play a role in platelet adhesion, aggregation, thrombogenesis and dermal connective tissue remodelling. In spite of some attempts towards compensatory angiogenesis in SSc, as evidenced by beta3 integrin subunit expression, it was evident that the angiogenic response was not able to prevent the development of hypovascularity during the advanced stages of the disease.

Raynaud's phenomenon following long-term repeated action of great differences of temperature.

The aim of these investigations was to study the effects of chronic thermal trauma on the development of vasomotor disturbances of the hands. The investigations were performed on 597 workers of the Fishing Company. The incidence of vasomotor disturbances of the hands was compared in: (1) workers not exposed to thermal trauma; (2) workers with long-term exposure to cold; (3) workers exposed to alternating influence of cold and heat. Superficial temperature was determined, finger plethysmography, capillaroscopy and hand arteriography were carried out. The investigations demonstrated that long-term alternating exposure to thermal trauma causes development of vasomotor disturbances. Clinical manifestations of Raynaud's syndrome were found in nearly 50% of female workers in fish processing plant whose hands were exposed to the action of ice and hot water. The incidence of vasomotor disturbances in workers exposed to long-term effects of cold was low.

Experience with Doppler investigations in vibration syndrome.

The aim of this study was to examine the possibility of applying Doppler flowmetry in diagnosing vascular changes in workers exposed to vibration. Polish, directional, continuous-wave Doppler apparatus UDP-10, has been used for evaluation of multisegmental blood pressure, velocity flow tracing and for making an ultrasonic "map" of the upper limbs. The thermal test used in this investigation allowed us to distinguish functional and organic alterations in the hand arteries. The present study has been performed in 113 workers, employed in an iron casting company and exposed to vibration. The control group comprised 62 healthy men. Raynaud's phenomenon has been found in 34 workers. It has been confirmed that Doppler equipment is a safe, noninvasive technique, with a high percentage of diagnostic accuracy and is a great aid in establishing the severity of symptoms in patients with vasospastic disease. As a result of our study the diagram for diagnosing patients with vasospastic disease has been elaborated and presented in this work.

Surgical management of abdominal aortic aneurysms in Poland. A multi-centre study.

Abdominal aortic aneurysm resections were performed on 941 patients between 1987 and 1991 in nine selected university vascular units in Poland. The aim of the study was (1) to determine how grave the problem of abdominal aortic aneurysms is in the main vascular centres in our country, (2) to evaluate the methods of management, (3) to trace the most common postoperative complications, and (4) to estimate results. Hospital mortality rate for 730 elective and urgent resections was 8.2%. The emergency resection mortality rate for ruptured aneurysm was 60.2%. The most common postoperative general complications were: cardiac (178-18.9%), pulmonary (76-8.1%), renal failure (58-6.2%) and cerebrovascular accidents (23-2.4%). The postoperative local complications (113) occurred in 87 (9.2%) patients. The most common were: colon ischemia (22-3.5%), haemorrhage (30-3.2%), acute graft occlusion (22-2.3) and peripheral embolism (19-2%). Sixty-five patients required early reoperation undergoing a total of 74 additional operative procedures. The local complications occurring in analysed material significantly influenced the results. Mortality in reoperated patients was almost twice as high as among those not reoperated (p < 0.01). Analysis of the material revealed no differences in the obtained results of aneurysm surgery in the succeeding years of our study, when expecting improvement in the last years. The cause of this could be treatment of more high risk patients. The absolute number of patients with abdominal aortic aneurysms referred to the unit influenced results.(ABSTRACT TRUNCATED AT 250 WORDS)

Retroperitoneal rupture of abdominal aortic aneurysms.

OBJECTIVE: About 40% of patients with ruptured abdominal aortic aneurysm (AAA) die before admission to the hospital. The next 40-50% of patients who reach a hospital die in the perioperative period or within 30 days after surgery. Two groups of patients with ruptured AAA can be distinguished: first-with intra-abdominal rupture, second-with retro-peritoneal rupture. The aim of the study was a retrospective analysis of the treatment results in patients with retro-peritoneal rupture of AAA. MATERIAL AND METHODS: 78 patients underwent a surgical procedure between 1.01.1985 and 30.10.1996. 78 patients (68 men and 10 women), mean age 67.6 (53-94) were included in this study. Based on diagnostic and surgical procedures, two periods of treatment can be distinguished. In the first period of the time from 1.01.1985 to 31.12.1992 patients were operated on immediately after admission to the hospital. In the second period from 1.01.1993 to 31.10.1996 patients were admitted to Intensive Care Unit. In this unit patients were intensively treated and prepared for surgical procedure. During this time the computerized tomography scanning (CT) was performed. RESULTS: In the first period the perioperative mortality was 75%. In the second period the perioperative mortality was 41.3%. DISCUSSION: The 1.5-2 hours postponement of operative procedure of the patients with retro-peritoneal rupture of AAA can decrease perioperative mortality. During the time patients were intensively treated and prepared for surgical procedure and CT examination enabled to choose proper surgical technique.