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Pituitary adenylate cyclase activating polypeptide is a potent vasodilator and oedema potentiator in rabbit skin in vivo.

1. The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on microvascular blood flow and plasma protein leakage were investigated in rabbit skin in vivo. 2. Intradermal injection of PACAP38, the 38 amino acid form of the peptide, caused a dose-dependent increase in blood flow measured by a 133Xe clearance technique. An equivalent increase in blood flow was induced by 10(-12) mol per site of PACAP38, 10(-12) mol per site of human alpha-calcitonin gene-related peptide (CGRP) and 10(-10) mol per site of vasoactive intestinal polypeptide (VIP). 3. The vasodilator activity of PACAP38 was not significantly different from that of the 27 amino acid form of the peptide, PACAP27, when measured with a laser Doppler flow meter, causing a 104 +/- 14% compared with 110 +/- 18% increase above basal blood flow at 10(-12) mol per site respectively. 4. At 10(-12) mol per site the effect of PACAP38 was longer lasting than that of CGRP. Blood flow remained significantly increased above control at 2 h with PACAP38 (P less than 0.05) whereas blood flow after intradermal CGRP had returned to control values by this time. 5. PACAP38 injected alone had no significant effect on microvascular leakage of 125I-labelled albumin. However, PACAP38 significantly potentiated bradykinin-induced oedema where it was approximately 100 fold more potent than VIP. 6. Oedema potentiation induced by PACAP38 was not inhibited by indomethacin at a dose which did inhibit potentiation of bradykinin-induced oedema by arachidonic acid.7. PACAP38 is at least as potent as other peptides which have been postulated to be involved in the inflammatory response when tested in rabbit skin in vivo. PACAP may contribute to both the hyperaemia and oedema components of inflammation.

Animals↗

Increased plasma level of T-kininogen in rats treated with Freund's adjuvant.

Studies have been carried out to clarify which component of plasma kininogen in rats increased in the inflammatory condition induced by an injection of Freund's complete adjuvant. Plasma T-kininogen, which was measured by assaying the amount of T-kinin liberated by trypsin treatment, remarkably increased in parallel with the severity of paw swelling following the intradermal injection of adjuvant into the rat hindpaw. Treatment with indomethacin or dexamethasone following an injection of adjuvant suppressed the increase in T-kininogen level as well as the development of paw swelling in rats. These results indicate that T-kininogen, the newly found precursor of T-kinin, is the main component of plasma kininogen which responds to the inflammatory stimulus in adjuvant arthritis.

Animals↗

Hyperbaric oxygen effects on brown recluse spider (Loxosceles reclusa) envenomation in rabbits.

Human loxoscelism was modeled in albino rabbits by injection of brown recluse spider (Loxosceles reclusa) venom, and the effects of daily or twice-daily hyperbaric oxygen treatment on wound healing were investigated. Lesions similar to those seen in humans were produced in rabbits by intradermal injection of 200 microliters of a venom extract (0.21 microgram protein per microliter), including edema and erythema, ischemia and cyanosis in the first 12 hr, extensive purpura by 24 hr, and crateriform ulcer formation by day four, with induration and eschar formation. Hyperbaric oxygen treatments, consisting of two atmospheres absolute (2 ATA) for 60 min, were applied daily (n = 8) or twice daily (n = 8), while control animals (n = 8) received no treatment. Treatments were initiated 72 hr after venom injection (day 3) to duplicate typical clinical treatment delays, and were administered for seven consecutive days. No significant effects of hyperbaric oxygen treatment on lesion healing were seen as measured by lesion area. However, histologic evaluation of wound tissue collected at euthanasia on day 24 showed clear differences between rabbits receiving twice-daily treatments and those receiving daily or no treatment. The former showed complete re-epithelization or slight ulceration, while the latter usually had necrotic cavities extending into the dermis, with myonecrosis and inflammatory cell accumulation. Thus, no superficial differences were seen between groups, but twice-daily treatments resulted in enhanced recovery at the histologic level.

Animals↗

Secondary hyperalgesia to mechanical but not heat stimuli following a capsaicin injection in hairy skin.

A psychophysical investigation was carried out to examine whether heat hyperalgesia exists within the secondary mechanical hyperalgesia zone surrounding a capsaicin injection site on hairy skin. A non-contact laser stimulator was used to deliver temperature controlled stimuli to sites within and outside the zone of mechanical hyperalgesia. Heat testing was carried out before and after the intradermal injection of 50 micrograms of capsaicin into the volar forearm. The zones of mechanical hyperalgesia to punctate and stroking stimuli and the region of flare were also mapped after the capsaicin injection. Heat pain thresholds inside the secondary mechanical hyperalgesic zone were not significantly different from thresholds outside the secondary mechanical hyperalgesia zone. In addition, pain ratings to an ascending series of heat stimuli delivered inside the zone of secondary hyperalgesia were not significantly different from pain ratings outside the zone of secondary hyperalgesia. Thus, there was no evidence for heat hyperalgesia within the zone of secondary hyperalgesia to punctate mechanical stimuli. Though the areas of punctate and stroking hyperalgesia were correlated, no correlation existed between the magnitude of capsaicin evoked pain and the areas mechanical hyperalgesia to punctuate and stroking stimuli or the area of flare. This suggests that independent mechanisms may mediate evoked pain, central sensitization that leads to mechanical hyperalgesia, and axon reflexive flare.

Analysis of Variance↗

alpha(4) integrin-dependent eosinophil recruitment in allergic but not non-allergic inflammation.

1. Although anti-alpha(4) integrin mAbs reduce eosinophil accumulation in several models of allergic inflammation, it is not clear whether this occurs via a direct action to block eosinophil alpha(4) integrins or indirectly on another cell type. The role of alpha(4) integrins on the accumulation of (111)In-labelled eosinophils in allergic and non-allergic inflammation in guinea-pig skin was therefore investigated. 2. Intradermal injection of antigen in sensitized skin sites induced accumulation of (111)In-eosinophils that was reduced up to 70% by two anti-alpha(4) integrin mAbs. In contrast, accumulation of (111)In-eosinophils to intradermal chemoattractants was unaffected by the same mAbs. 3. Accumulation of (111)In-eosinophils in allergic and non-allergic conditions was partly inhibited by a low dose of an anti-beta(2) integrin mAb. In combination with anti-alpha(4) integrin mAb, responses were not further reduced suggesting that these adhesion pathways are not additive or synergic. 4. Pretreating skin sites with antiserum or contaminating LPS did not reveal an alpha(4) integrin dependent pathway for chemoattractant-induced (111)In-eosinophil accumulation. These data suggest that alpha(4) integrins are involved in the response to antigen in sensitized skin sites. 5. Pretreating (111)In-eosinophil with alpha(4) integrin mAb blocked their adhesion to fibronectin in vitro but did not inhibit their accumulation in allergic inflammation suggesting that the blocking effect in vivo was eosinophil independent. 6. These data support the concept that targeting alpha(4) integrins on cells other than eosinophils could control eosinophil accumulation and have therapeutic potential in allergic diseases such as asthma and atopic dermatitis.

Animals↗

Role of adrenergic stimuli in parathyroid hormone secretion in man.

The role of adrenergic stimuli in the secretion of parathyroid hormone (PTH) in man was evaluated. Intradermal injections of isoproterenol, 0.15 mg, or epinephrine, 0.3 mg, caused significant prompt increases in serum PTH levels. These increases were not accompanied by any changes in serum calcium (Ca) during the period of observation. Phenylephrine, 1.5 mg, intradermally, did not cause any significant changes in serum PTH or serum Ca. Propranolol infusion alone significantly inhibited the basal secretion of PTH. This inhibition by propranolol was overcome by isoproterenol administration. The results indicate that 1) beta adrenergic agents increase PTH secretion whereas alpha adrenergic agents have no effect, 2) beta adrenergic stimuli probably play an important physiological role in basal PTH secretion in man.

Adrenergic Agonists↗

Bronchopulmonary macrophage activation in the pathogenesis of hypersensitivity pneumonitis.

Repeated intratracheal (IT) inoculation of rabbits with a homogenized, saline suspension of Micropolspora faeni produced bronchopulmonary (BP) histologic lesions resembling those of human hypersensitivity pneumonitis. With an in vitro phagocytic and bactericidal assay, an analysis of BP macrophages from M. faeni-injected rabbits demonstrated activation at both 2 and 4 weeks after the initiation of immunization. No BP macrophage activation was observed in immunized rabbits 6 weeks post-inoculation. BP macrophage activation was capable of recall after 6 weeks in M. faeni-sensitized animals that received a booster IT injection (2 mg) that did not activate "normal" alveolar wash cells. This recall of BP macrophage activation was accompanied by both a marked migration of mononuclear cells into the lung and positive delayed hypersensitivity skin reactions after intradermal injection of M. faeni antigen. Pulmonary histologic examination of sensitized, boosted rabbits suggested an enhanced cellular parenchymal infiltrate when compared with appropriate controls. The above observations confirm the occurrence of immunologically activated BP macrophages in rabbits inoculated witn M. faeni via the respiratory tract route and suggest a correlation between macrophage activation and histopathology.

Actinomycetales↗

[Experimental autoimmune uveo-retinitis (EAU): cellular immunity and humoral antibodies in the induction of ocular lesions and tolerance to retinal antigens].

EAU is induced in guinea pigs by one inoculation into the foot-pad of a retinal extract in complete Freund's adjuvant. The evolution of the antibodies (complement fixation) is compared with the évolution of the disease (intensity evaluated by frequent ophthalmological examinations). The successful passive transfer of the disease by local injections of immune serum and the demonstration by immunofluorescence of immune complexes in the ocular tissues are arguments for the role of humoral antibodies in the induction of the disease. EAUmay be inhibited by repeated subcutaneous or intradermal injections of the antigen, either before or after the onset of the inflammation. These injections depress the delayed hypersensitivity and increase the titre of complement fixing and passive anaphylactic antibodies.

Animals↗

Eosinophil infiltration into human skin is antigen-dependent in the late-phase reaction.

Eosinophils play a critical role in late-phase reaction allergic inflammatory responses, although the factors responsible for selective tissue eosinophilia are currently ill-defined. To determine whether recruitment of eosinophils is allergen-specific, or a feature of inflammation in allergic individuals, we have examined cutaneous cell infiltrates and endothelial cell adhesion molecule expression in atopic subjects 6 h (n = 8) and 24 h (n = 7) following ultraviolet-B (UVB) irradiation, or intradermal injection of late-phase reaction allergens or diluent control, using standard immunohistochemical techniques. The numbers of eosinophils were increased significantly, when compared to controls, at both 6 h (P < 0.01) and 24 h (P < 0.05), following intradermal allergen challenge, whereas no significant increase in eosinophils was observed following UVB irradiation. UVB and allergen both induced significant increases in neutrophils, monocytes and T cells at 24 h compared to control sites. An increased expression of endothelial cell adhesion molecules, E-selectin and intercellular adhesion molecule-1 (ICAM-1), was observed in both models of inflammation. Vascular cell adhesion molecule-1 (VCAM-1) was induced weakly on some biopsies following allergen, and not at all following UVB. These data indicate that eosinophil infiltration in susceptible individuals is a specific property of allergen. Although this study would support the postulated role of VCAM-1 in selective eosinophil recruitment, given its variable and weak expression, additional factors are likely to be involved.

Allergens↗

Acetylcholine sweatspot test for autonomic denervation.

A test for autonomic denervation based on the local sweat response to 0.1 ml 1% acetylcholine administered intradermally, which depends on an intact local sympathetic supply, is described. Diabetic autonomic neuropathy affects the longest fibres first and thus the test was applied to the feet. After painting a standard site on the dorsum of the foot with iodine and starch, acetylcholine was injected intradermally in the centre. The normal response, visible to the eye, is a uniform distribution of dark spots of iodine discolouration at the sites of sweat production. In diabetic autonomic neuropathy this pattern is lost to a varying degree. In a photographic image magnified x 10, the spots were counted in sixty 2.5 cm squares in a grid centred on the injection site. 50 normal volunteers aged 18-69 were tested. No effect of age or sex was found. Five or more squares with less than 6 spots was the definition of abnormal. 24 diabetic men who complained of impotence were investigated with the sweatspot test, a pupil test, and cardiovascular autonomic function tests. 13 had abnormal sweatspot tests with scores up to sixty squares with less than 6 spots. In keeping with the increased length of the sympathetic fibres to the feet compared with those to the iris, there was a 30% false-negative rate for the pupil test if the sweatspot test is taken as standard. Agreement between the cardiovascular tests and the sweatspot test was seen in only 17 patients. The sweatspot test appears to be a more sensitive indicator of autonomic neuropathy than the commonly used cardiovascular tests.

Acetylcholine↗

Sentinel node localization in primary melanoma: preoperative dynamic lymphoscintigraphy, intraoperative gamma probe, and vital dye guidance.

BACKGROUND: Sentinel node (SN) biopsy can be used to select patients with primary melanoma for therapeutic lymphadenectomy. The aim of the study was to assess the efficacy of 3 methods to locate the SN: preoperative dynamic lymphoscintigraphy, intraoperative patent blue dye (PBD), and gamma-detecting probe (GDP). METHODS: We studied 133 patients with cutaneous melanoma and clinically negative lymph nodes. Within 24 hours before surgery, colloid labeled with technetium 99m was injected intradermally around the site of the primary melanoma. The patients were studied before their operations by using dynamic lymphoscintigraphy. A total of 208 SNs were found in 164 lymph node basins. In addition, all the patients had PBD injected immediately before the surgical procedure. When the blue-stained node was identified intraoperatively, its radioactivity level was measured with the GDP. In the absence of blue coloration, the GDP was used to trace the SN. RESULTS: Of 208 SNs, 168 (80.8%) were identified in the regional draining basin during intraoperative lymphatic mapping by using PBD. By using the GDP method, 202 (97.1%) of 208 were identified (GDP vs PBD; P < .01). By combining the 2 methods, 206 (99%) of 208 SNs were detected. Of the 133 patients, 29 (21.8%) had pathologically positive SNs, and were subsequently subjected to regional lymphadenectomy. In 26 (89.7%) of 29 patients, the SN was the only node with metastasis. Three cases (10.3%) of recurrence in patients with microscopic SN metastasis and 7 cases (6.7%) of recurrence in patients without SN metastasis were found during a median follow-up of 566 days. CONCLUSIONS: Preoperative dynamic lymphoscintigraphy and intraoperative mapping with PBD and GDP offer simple and reliable methods of staging regional lymph nodes without subjecting every patient to a regional lymphadenectomy.

Adolescent↗

Antitumour effect of a synthetic analogue of fumagillin on murine renal carcinoma.

OBJECTIVE: To evaluate the antitumour effect of an angio-inhibitory drug, a synthetic analogue of fumagillin (TNP-470), on murine renal carcinoma (Renca) in vivo and in vitro. MATERIALS AND METHODS: The effect of TNP-470 on the growth of Renca cells in vitro was measured by angiogenesis assay and cell counting with dye exclusion. In the angiogenesis assay, Renca cells were injected intradermally and the number of blood vessels orientated towards the tumours was counted 3 days after tumour inoculation. To examine the effect of TNP-470 on the subcutaneous tumour growth and lung metastasis of Renca, Renca cells were injected subcutaneously or intravenously in BALB/c mice and they were treated with a subcutaneous injection every 3 days. RESULTS: Dose-dependent growth inhibition in vitro was observed with 50% inhibition occurring at 600 ng/ml. Angiogenesis assay revealed that administration of TNP-470 inhibited the angiogenesis induced by Renca in a dose-dependent manner. In the subcutaneous experiment, TNP-470 decreased the growth rate of established subcutaneous tumours rather than reduced the size of the tumour. The administration of TNP-470 in mice with lung metastasis inhibited the development of metastasis of Renca without weight loss or diarrhoea. CONCLUSION: The present study demonstrated that TNP-470 had an inhibitory effect on tumour-induced angiogenesis and a significant anti-tumour effect on Renca. This suggests that TNP-470 could be useful in the treatment of renal cell carcinoma. Further studies are needed to clarify whether TNP-470 is more effective when combined with other drugs such as interferons.

Animals↗

Glucans and dextrans in rats and man.

Intracutaneous injections of three glucan contaminants of invert sugar solutions and crude cane sugar into human skin produced localised wheals and erythema reactions. These glucans of molecular weight between 12,000 and 1,400,000 were also active on intradermal injection into both dextran-sensitive and dextran-resistant rats, and, like dextrans, were active on subcutaneous injection into dextran-sensitive animals.

Animals↗

Therapeutic effects of Hominis placenta injection into an acupuncture point on the inflammatory responses in subchondral bone region of adjuvant-induced polyarthritic rat.

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial membrane in the joint, which leads to the progressive destruction of articular cartilage, ligament and bone. Several cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and interleukin 6 (IL-6) have been implicated in the pathological mechanisms of synovial tissue proliferation, joint destruction and programmed cell death in rheumatoid joint. In the Korean traditional medicine, Hominis placenta (HP) as an herbal component of herb-acupuncture has been widely used to treat chronic inflammatory diseases such as RA. To study the therapeutic effects of HP injection into the ST36 acupoint (HP herb-acupuncture) on the inflammatory responses of a subchondral region of rheumatoid joint, the polyarthritis-induced Sprague-Dawley (SD) rat was developed as a rheumatoid arthritis model by the intradermal injection of dried cells of Mycobacterium tuberculosis emulsified in squalene to the base of tail. After the onset stage (11 d after adjuvant injection) of polyarthritis, a fixed volume of HP extract was daily injected to Zusanli (ST36) acupoint on the rat's leg for 2 weeks. The body weight, paw volume of the knee joint and articular index were exploited as an assessment method addressing arthritic symptoms, and the expression profiles of TNF-alpha, IL-1beta and IL-6 at the subchondral bone of the joint were analyzed using an immunohistochemistry. After the treatment of arthritic rats with HP, the body weights and paw volumes of arthritic rats were almost restored to the levels of normal rats whereas the evaluation by the articular index was not remarkable. The TNF-alpha, IL-1beta and IL-6 positive cells in the immunohistological sections of subchondral bone region of the joint significantly decreased in HP-treated (ST36 acupoint) arthritic group as compared with those in non-treated or HP-treated (non-acupoint) ones, which was coincident with the behavioral studies. In conclusion, the HP herb-acupuncture was found to be effective to alleviate the arthritic symptoms in adjuvant-induced arthritis rats as regards the body weight, joint appearance and the expression profiles of inflammatory cytokines.

Acupuncture Points↗

[Delayed hypersensitivity to Trypanosoma cruzi antigen. I -- Experimental study in rabbits].

Delayed-type skin hypersensitivity in rabbits infected with homologous or heterologous T. cruzi has been elicited upon injection of 50 micrograms of protein in the T12E antigen derived from a parasite clone. The specificity of this reaction was indicated by absence of skin reactivity in control rabbits that received same quantity of antigen. Also, the intradermal injection of the antigen in five occasions in control rabbits neither induced seroconversion nor shifted their skin sensitiveness. On the other hand, chagasic rabbits that underwent a series of five skin testings one week apart, did not alter the intensity of the skin reactivity, and their specific serum antibody titers remained similarly high. Of interest, the ECG patterns remained unchanged after a series of five skin testings in control and in chagasic rabbits. This study shows that the T12E antigen might be an useful tool for diagnosing T. cruzi infections, besides serving as an immunologic marker for morbidity in Chagas' disease.

Animals↗

Immunostimulatory DNA sequences influence the course of adjuvant arthritis.

Bacterial DNA is enriched in unmethylated CpG motifs that have been shown to activate the innate immune system. These immunostimulatory DNA sequences (ISS) induce inflammation when injected directly into joints. However, the role of bacterial DNA in systemic arthritis is not known. The purpose of the present experiments was to determine whether ISS contributes to the development of adjuvant arthritis in Lewis rats after intradermal injection of heat-killed Mycobacterium tuberculosis (Mtb). The results showed that Mtb DNA was necessary for maximal joint inflammation in adjuvant arthritis but could be replaced by synthetic ISS oligodeoxynucleotides. The arthritis-promoting effect of the Mtb DNA or of the ISS oligodeoxynucleotides correlated with an increased Th1 response to Mtb Ags, as measured by the production of IFN-gamma and increased production of the osteoclast differentiation factor, receptor activator of NF-kappaB ligand (RANKL). The Mtb DNA did not enter the joints but dispersed to the bone marrow and spleen before the onset of systemic joint inflammation. Thus, adjuvant arthritis is a microbial DNA-dependent disease. In this model, we postulate that massive and prolonged activation of macrophages, dendritic cells, and osteoclast precursors in the bone marrow may prime the joints for the induction of inflammatory Th1 immune responses to Mtb Ags.

Adjuvants, Immunologic↗

Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer: a phase I/II clinical trial.

INTRODUCTION AND OBJECTIVES: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor-specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by 'naked' DNA immunization or by injecting replication-deficient viral vectors that carry the tumor-specific DNA. Using the prostate- specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer. The primary objective of the study was to determine the safety of the PSMA vaccine after repeated intradermal injections. METHODS: We have included the extracellular human PSMA DNA as well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette from the PSMA plasmid was inserted into a replication deficient adenoviral expression vector. Twenty-six patients with prostate cancer were entered into a phase I/II toxicity-dose escalation study, which was initiated in spring 1998. Immunizations were performed intradermally at weekly intervals. Doses of DNA between 100 and 800 microg and of recombinant virus at 5x10(8) PFUs per application were used. RESULTS AND CONCLUSION: No immediate or long-term side effects following immunizations have been recorded. All patients who received initial inoculation with the viral vector followed by PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed-type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of the patients who received a PSMA plasmid and CD86 plasmid, only 50% showed signs of successful immunization. Of the patients who received PSMA plasmid and soluble GM-CSF, 67% were immunized. However, all patients who received the PSMA/CD86 plasmid and sGM-CSF became immunized. The patients who did not immunize during the first round were later successfully immunized after a boost with the viral vector. The heterogeneity of the medical status and the presence in many patients of concomitant hormone therapy does not permit unequivocal interpretation of the data with respect to the effectiveness of the therapy. However, several responders, as evidenced by a change in the local disease, distant metastases, and PSA levels, can be identified. A phase II clinical study to evaluate the effectiveness of the therapy is currently underway.

Adenoviridae↗

[Differences in antitumor effect of various BRMs by intratumoral administration: induction of immunosuppressive acidic protein].

The antitumor effects of biological response modifiers (BRM) in a new experimental mouse model, the "double grafted tumor system", were analysed. BALB/c mice received simultaneous inoculations of Meth-A fibrosarcoma cells on right flank (10(6) cells) and left flank (2 x 10(5) cells) on day 0, and BRMs were injected intratumorally into right tumor on day 3, 4 and 5. The growth of the left-flank tumor was the real target for the evaluation of a given drug after 21 days. PSK (a protein-bound polysaccharide preparation), IL-1 and Cepharanthin, cured not only the right, but also the left, non-treated tumor in a double grafted tumor system. OK-432 (a Streptococcus preparation) and BCG cured the right tumor and inhibited the growth of the left tumor. Lentinan (a polysaccharide preparation) inhibited neither the right nor the left tumor. Immunosuppressive acidic protein (IAP) in serum was increased transiently soon after intradermal injection of PSK, OK-432 and TNF in BALB/c mice. But Lentinan did not induce IAP. IAP in serum was gradually increased after intradermal inoculation of Meth-A tumor in BALB/c mice. At 21 days after tumor inoculation, IAP in serum reached a maximum level (300 micrograms/ml). The serum IAP level of Meth-A-bearing mice as well as that of normal mice increased after the intratumoral injection of PSK. At 21 days after tumor inoculation, IAP in PSK-treated mice returned to normal level. The biochemical differences between PSK-induced IAP (early, inflammatory IAP) and Meth-A-induced IAP (late, tumor-induced IAP) was investigated by crossed immunoaffino electrophoresis (CIAE). Inflammatory IAP was rich in biantennary sugar chain, and tumor-induced IAP was rich in tri-tetraantennary sugar chain.

Animals↗