Therapeutic potentials of bovine colostrums.

Colostrum is the first milk produced by mammals for their young ones. This transfers the passive immunity gained by the mother to the baby. The bovine colostrum (BC) can be obtained in large quantity and has properties similar to human colostrum. It has been used for various disorders of the body. It has properties to stimulate immune system, contains growth factors and many bioactive substances needed for the body to combat with wear and tear. The BC has been used for various gastrointestinal disorders, respiratory tract infection, rheumatoid arthritis, healing injured tissues of body etc. There are not much double blind placebo-controlled trials to prove its efficacy, though a lot of experience about its good effects in various disorders is available in the literature. The dosage and duration of therapy need to be worked up. The BC has potential to treat as well to prevent certain diseases in the body. In future this will prove to be a very useful product to treat and control diseases in a natural way.

Functional improvement by electro-acupuncture after transient middle cerebral artery occlusion in rats.

Functional recovery by the application of electro-acupuncture (EA) on different acupoints was investigated using a transient middle cerebral artery occlusion (MCAO) model in rat. Acupoints were Baihui (D20) plus Renzhong (D26) (MCAO + D group), and Hanyan (G4), Xuanlu (G5), Xuanli (G6), plus Qubin (G7) (MCAP + G group). Animals with EA treatment showed significant functional improvements from 12 days after the reperfusion against those without EA treatment. Among EA treated groups, MCAO + G showed a more significant recovery than MCAO + D. Infarct volume revealed the significant reduction in the EA treated groups especially in MCAO + G at 30 days. Immunohistochemical study showed a remarkable induction of vascular endothelial growth factor (VEGF) in astrocytes of the peri-infarct area at 30 days, more in EA treated groups than in groups treated with MCAO alone. These results suggest that the acupoints applied in this study are effective for the functional recovery, and an enhanced expression of VEGF may play a certain role in recovery process after stroke.

[Mechanisms of the protective action of plant preparation erakond in influenza]. / Mekhanizmy protektivnogo deistviia fitopreparata érakond pri grippe.

The results of the study of some mechanisms of the protective action of Erakond, a phytopreparation intended for use in acute influenza infection are presented. In controlled animal observations Erakond was shown to induce the synthesis of antibodies in infected and immunized mice, the production of lymphokine, endogenic interferon; to increase the phagocytic and adhesive activity of mononuclears. The preparation facilitated the normalization of the process of the peroxide oxidation of lipids in mice and rendered a positive effect on the course of the inflammatory process in the lungs of mice at the final stage of influenza infection.

Possible involvement of the vascular endothelial growth factor-Flt-1-focal adhesion kinase pathway in chemotaxis and the cell proliferation of osteoclast precursor cells in arthritic joints.

Vascular endothelial growth factor (VEGF) plays a crucial role in the pathogenesis of inflammatory joint disease, including angiogenesis and synovitis. Rheumatoid arthritis is a chronic inflammatory disease characterized by progressive synovitis and subsequent bone destruction mediated by osteoclasts (OCs). In this study, we investigate the effects of VEGF on OC precursor cells (pOCs) using Raw cells and adjuvant-induced arthritis in rats. OCs and pOCs in the arthritic joints express VEGF and VEGF receptor type I (Flt-1). Raw cells also express Flt-1, and VEGF treatment stimulated chemotaxis, cell proliferation, the association of Flt-1 with focal adhesion kinase (FAK), and the tyrosine phosphorylation of FAK in Raw cells. The tyrosine phosphorylation of FAK was also observed in pOCs in the arthritic joints of adjuvant-induced arthritis. Adenovirus-mediated expression of FAK-related nonkinase in Raw cells inhibited the effects of VEGF in a dominant negative manner. Furthermore, intra-articular injection of the FAK-related nonkinase virus suppressed the recruitment of pOCs and bone destruction. Our results suggest the possible involvement of the VEGF-Flt-1-FAK pathway in inflammatory disease-induced joint destruction.

Matrix metalloproteinase 9, apoptosis, and vascular morphology in early arthritis.

OBJECTIVE: To examine matrix metalloproteinase 9 (MMP-9) in the synovial fluid (SF) and synovial membrane (SM) in relation to vascular endothelial cell (EC) apoptosis, vascular endothelial growth factor (VEGF), and SM vascular pattern. METHODS: Thirty-four patients underwent needle arthroscopy of the knee joint; 12 had early rheumatoid arthritis (RA), 12 had early psoriatic arthritis (PsA), and 10 had osteoarthritis (OA). The early RA and early PsA patients were matched for disease activity. SF levels of MMP-9 and VEGF were measured by an enzyme-linked immunosorbent assay, and EC apoptosis was measured by TUNEL assay. MMP-9 expression was examined in SM by immunohistochemistry. Synovial tissue explants were stimulated with VEGF, and MMP-9 levels were measured in the supernatants. The synovial vascular pattern was recorded. RESULTS: SF MMP-9 levels were significantly higher in early PsA patients than in early RA patients; OA patients had minimal levels. MMP-9 levels correlated with blood vessel morphology and SF VEGF levels. MMP-9 expression was greater in early PsA SM than in early RA SM, but the difference was not significant. In contrast however, EC apoptosis was greater in early RA SM than in early PsA SM. MMP-9 levels increased 2-fold and 9-fold, respectively, in SM explant culture supernatants on day 7 in response to stimulation with 25 ng/ml and 50 ng/ml of VEGF. CONCLUSION: SF MMP-9 levels correlate with the pattern of SM neovascularization and SF VEGF levels in early inflammatory arthritis, and VEGF increases MMP-9 production by SM. Endothelial cell apoptosis, however, appears to be more prevalent in early RA. This combination of factors may explain the pattern of differential angiogenesis in these arthritides.

Unbound vascular endothelial growth factor and its receptors in breast, human milk, and newborn intestine.

BACKGROUND: Human milk, rich in cytokines, may contain the potent permeability- and angiogenesis-promoting agent vascular endothelial growth factor (VEGF). OBJECTIVE: We wanted to study whether free or bound VEGF is present in human milk and whether it and its receptors (VEGFR-1 and -2) are expressed in lactating breast or newborn intestinal tissue. DESIGN: The study had a longitudinal design with collection of human milk from healthy (n = 32) and diabetic (n = 5) women at 2, 7, and 30 d postpartum. Milk was analyzed for VEGF by enzyme-linked immunosorbent assay along with plasma samples collected 2 d postpartum. Immunohistochemistry was used to localize VEGF and its receptors in lactating breast and newborn intestine. Gel filtration with radiolabeled VEGF was performed to study whether human milk contains VEGF binding proteins. RESULTS: Human milk VEGF concentrations in healthy (76 +/- 19 microg/L, x +/- SD) and diabetic (75 +/- 25 microg/L) women did not differ at 2, 7 (23 +/- 7 and 27 +/- 8 microg/L, respectively), or 30 d (14 +/- 5 and 17 +/- 7 microg/L, respectively) postpartum. VEGF was undetectable in all but 3 plasma samples. Human milk was free of VEGF binding proteins. VEGFR-1 and -2 immunoreactivity was seen in the glandular epithelial cells of the newborn intestine and lactating breast, whereas VEGF was present only in breast glandular epithelium. CONCLUSIONS: The high concentrations of VEGF in human milk, especially colostrum, are not affected by maternal diabetes and may play a role in newborn nutrition.

Modulation of murine mammary tumor vasculature by dietary n-3 fatty acids in fish oil.

We have previously shown that mice fed a high (n-3) fatty acid-containing diet with 20% (w/w) total fat had significantly slower mammary tumor growth, decreased numbers of metastatic pulmonary nodules, and decreased total metastatic load. In this study we sought to determine whether tumor vascularization was altered in mice fed diets varying in concentrations of (n-3) and (n-6) fatty acids. Several direct or indirect parameters of vascularization were tested. With 20% dietary fat, fish oil (FO) or a mixture of FO and safflower oil (FS) significantly reduced blood vascular area, mast cell number and macrophage infiltration in solid mammary tumors compared to tumors grown in mice fed safflower oil (SO). A decreasing trend was seen in the percent area of vessels positive for CD31 and vascular endothelial growth factor (VEGF) in the 20% FO and 20% FS compared to the 20% SO dietary groups. VEGF concentrations were twice as high in smaller tumors (100 mm3) from all dietary groups as compared to larger tumors (500 mm3). A two-fold increase in VEGF levels was found in the 20% SO dietary group compared to the 20% FO group in 100-mm3 but not larger tumors. We conclude that at 20% total fat, the n-3 fatty acids found in fish oil may inhibit primary mammary tumor growth through modulation of select determinants of vascularization.

Tumour necrosis factor-alpha is involved in the contrasting effects of ultraviolet B and ultraviolet A1 radiation on the release by normal human keratinocytes of vascular permeability factor.

Erythema and the initiation of an inflammatory response are typical features of human skin after ultraviolet (UV) radiation (UVR) exposure. Among the soluble factors that account for the induction of an erythema, the most recently discovered is vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a potent inducer of microvascular permeability which is expressed by keratinocytes. As epidermal cells are the first target cells of UVR, we studied the effects of UVBR (312 nm) and UVA1R (365 nm) on the secretion of VEGF by normal human keratinocytes and evaluated the role of interleukin 1 alpha (IL-1 alpha) and tumour necrosis factor-alpha (TNF-alpha) in this process. UVBR (100 and 200 mJ/cm2) induced a dose-dependent increase in the release by normal human keratinocytes of VEGF, which is widely mediated through the release of TNF-alpha but not IL-1 alpha. Conversely, UVA1R (5 and 7 J/cm2) did not modify the basal level of VEGF and did not induce the secretion of TNF-alpha by keratinocytes. Moreover UVA1R, when associated with UVBR, inhibited the increase in VEGF induced by UVBR alone. Taken together, these findings indicate that UVBR and UVA1R have a contrasting effect on the release of VEGF, which is widely mediated by TNF-alpha. They may partly explain the minor erythematous effect of UVA1R and its beneficial role in cutaneous phototherapy.

Increased levels of leukemia inhibitory factor in synovial fluid from patients with rheumatoid arthritis and other inflammatory arthritides.

OBJECTIVE: To examine synovial fluid (SF) from patients with arthritis, for the presence of the cytokine leukemia inhibitory factor (LIF). METHODS: SF from 152 subjects was examined for LIF, using a radioreceptor competition assay. RESULTS: LIF was present at concentrations of 1-43 ng/ml in the SF of 23% of patients with rheumatoid arthritis (RA) or other inflammatory or infectious arthritides but in only 1 of 29 patients with osteoarthritis (P < 0.01). In the RA patients, the SF LIF concentration correlated significantly with the peripheral blood white blood cell count (WBC) (P < 0.05) and the SF WBC count (P < 0.01), but not with other clinical or radiologic parameters of disease activity or progression. CONCLUSION: LIF is implicated as a potential mediator of the local or systemic inflammatory response or the joint destruction seen in inflammatory arthritis.

Studies on the allergenicity of the amino-terminal epitope (Bet v I 23-38) from birch pollen allergen.

An N-terminal peptide of the major allergen of birch (Bet v I 23-38) was selected for studying the activity of this segment on the basis of optimal hydrophilicity as it was tentatively suggested to be a surface exposed epitope. In addition two control peptides in the region 1-38 were similarly used for comparative assignment of the allergenicity. Peptide analogues from the amino acid terminal region, amino acid residues No. 23-38 of Bet v I, were synthesized by semiautomatic solid-phase peptide synthesis. In vitro and in vivo biological activity studies were performed on these analogous peptides. The IgE-binding capacity of the synthetic peptide 23-38 was examined using the following tests: specific IgE inhibition, skin prick test, nasal provocation and Prausnitz-Küstner inhibition. The results of these investigations suggested that the region 23-38 from the birch and hazel major allergen encompassed a single haptenic epitope.

Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies.

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.

A comparison of the antigenic and allergenic components of birch and alder pollens in Scandinavia and Australia.

Allergens in birch (Betula) pollens from B. pendula grown in Australia and Norway, B. davurica and B. populofolia and from alder (Alnus incana) were identified by electroblotting, following separation by SDS-PAGE, transfer to nitrocellulose membranes and incubation with sera from birch pollen-allergic subjects. Of 42 antigenic components detected by protein staining in the pollen extract from B. pendula grown in Norway, 17 bound IgE. The allergenic components included those already reported in the literature at MWs of 40, 29, 25, 17 and 10-12 kd, as well as previously undescribed components at MWs of 90, 79, 60, 50, 38, 35, 31, 27, 23, 16, 15 and 14 kd. The major IgE-binding components were located in the low MW region 10-17 kd for all species of birch and alder pollen proteins studied. Results reported here provide the first evidence of birch and alder pollen allergies in Australia. Extensive heterogeneity was observed amongst the sera from birch pollen-allergic subjects in both Norway and Australia. Cross-reactivity appears to exist among the proteins present in pollen from the various birch species and from alder.

Immunomodulation with Viscum album and Echinacea purpurea extracts.

Extracts of Viscum album (Plenosol) and Echinacea purpurea (Echinacin) are used clinically for their non-specific action on cell-mediated immunity. In vitro we could prove that these two extracts have a stimulating effect on the production of lymphokines by lymphocytes and in the transformation test. A toxic effect on cells was produced only with very high, clinically irrelevant concentrations. Clinical application of these extracts can produce a stimulation of cell-mediated immunity (one therapeutic administration followed by a free interval of one week) or can have a depressive action (daily administrations of higher doses). These observations were confirmed by lymphokine production and assay, 3H-thymidine incorporation and a skin test with recall antigens (Multitest Merieux).

Polyclonal B cell activation by a B cell differentiation factor, B151-TRF2. III. B151-TRF2 as a B cell differentiation factor closely associated with autoimmune disease.

We demonstrated previously that B151K12 T cell hybridoma produces two distinct B cell differentiation factors, B151-TRF1 and B151-TRF2, capable of inducing differentiation of antigen-activated and unstimulated B cells into antibody-forming cells, respectively. In the present study we investigated the pathophysiologic relation of these factors with factors obtained from MRL/MP-lpr/lpr(MRL/lpr) mice and (C57BL/6 X DBA/2)F1 (BDF1) mice undergoing chronic graft-vs-host reaction (GVHR), representing a murine model of systemic lupus erythematosus with polyclonal B cell activation associated with the T cell hyperfunction. The functional and biochemical analyses revealed that B151-TRF2-like, but not B151-TRF1-like, activity was found in culture fluid supernatant (CFS) of lymphoid cells from MRL/lpr mice with lymphoproliferative syndrome. On the other hand, both B151-TRF1- and B151-TRF2-like activities were detected in CFS prepared from spleen cells of BDF1 mice undergoing chronic GVHR by the inoculation of parental DBA/2 spleen cells. Interestingly, spleen cells of BDF1 mice transferred with DBA/2 thymocytes preferentially elaborated B151-TRF1-like factor. Because BDF1 mice transferred with DBA/2 spleen cells but not with DBA/2 thymocytes developed a SLE-like syndrome exemplified by the appearance of Coombs' antibody and proteinuria, it seemed likely that production of B151-TRF2-like factor was closely associated with the onset of autoimmune disease. In fact, B151-CFS containing B151-TRF2 but not B151-TRF1 activity could induce a striking autoantibody production both in vivo and in vitro as detected by PFC responses of normal mice to bromelain-treated mouse red blood cells (BrMRBC). Moreover, it was demonstrated that in vitro anti-BrMRBC PFC responses induced by semipurified B151-TRF2 was markedly inhibited by addition of relevant anti-Ia antibody to the culture. Thus, the present study demonstrates that B151-TRF2 represents one of the B cell differentiation factors responsible for polyclonal B cell activation leading to autoantibody production.

Presence of IgE suppressor factors in human colostrum.

In spite of intensive investigations, the ability of breast feeding to delay and to attenuate atopic diseases in children remains debatable. This study documents a mechanism whereby breast feeding might interfere with the synthesis of IgE by breast-fed infants. Indeed, we show that colostrum contains IgE-binding factors (IgE-BF) capable of suppressing the in vitro synthesis of human IgE. Colostrum obtained from 15 donors was successively depleted of lipids and casein, filtered through Amicon XM50 membrane (mol. mass cut-off 50 kDa) and lyophilized. IgE-BF was demonstrated in such preparations by two different approaches, i.e. a classical rosette inhibition assay and Western blot analysis. In the first instance, lyophilized preparations of colostrum inhibited the binding of IgE-coated bovine erythrocytes to IgE recovered on the surface of RPMI 8866 lymphoblastoid cells. The rosette-inhibiting activity could be absorbed on IgE- but not on IgG-Sepharose 4B and it could be recovered in the eluate of IgE-Sepharose 4B. The molecular mass of IgE-BF was comprised between 10 to 20 kDa as estimated by gel filtration through a calibrated Sephadex G-75 column. After fractionation on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose membrane, colostrum displayed one band of 14 kDa and reacted with radiolabeled IgE but not with IgG nor IgM. This 14-kDa band could be removed by absorbing colostrum with IgE- but not with IgG-Sepharose 4B. Most importantly, the colostrum IgE-BF suppressed the spontaneous in vitro synthesis of IgE by B lymphocytes derived from allergic donors without altering the production of IgM.

Modulation of the biologic activities of IgE-binding factors. I. Identification of glycosylation-inhibiting factor as a fragment of lipomodulin.

Splenic T lymphocytes of rats treated with complete Freund's adjuvant (CFA) spontaneously release a lymphokine that inhibits the glycosylation of IgE-binding factors during their biosynthesis and provides the factors with biologic activity: selective suppression of the IgE response. The lymphokine, which is called glycosylation-inhibiting factor, also prevents IgE-induced increases in Fc epsilon R+ cells. The glycosylation inhibiting factor is formed by stimulation of BCG-primed spleen cells with PPD and participates in the selective formation of IgE-suppressive factors. The lymphokine is derived from OX 8+ T cells in both the CFA and BCG systems. The glycosylation-inhibiting factor is a 16,000-dalton peptide, as estimated by gel filtration, and specifically binds to monoclonal antibody against lipomodulin, a phospholipase inhibitory protein. Furthermore, "lipomodulin" was detected by radioimmunoassay in the 16,000-dalton fraction that contained glycosylation inhibiting factor. The fraction did not inhibit phospholipase A2 but after alkaline phosphatase treatment, the fraction did inhibit phospholipase A2. Furthermore, purified lipomodulin obtained from glucocorticoid-treated rabbit neutrophils had the same biologic activities as glycosylation inhibiting factors; i.e., it inhibited both protein glycosylation of IgE-binding factors and IgE-induced expression of Fc epsilon R. The results collectively indicate that glycosylation-inhibiting factor is a fragment of phosphorylated lipomodulin.