Distant metastasis facilitated by BCG: spread of tumour cells injected in the BCG-primed site

Tumour metastasis in BCG-pretreated mice was studied using a methylcholanthrene-induced fibrosarcoma in C3H/He mice. When tumour cells were injected into the BCG-primed site, distant metastasis occurred in the lungs and the popliteal lymph node, through this tumour did not metastasize in normal mice. Such metastases were increased in proportion to the number of tumour cells injected into the BCG-primed site, and developed soon after tumour challenge. Concomitant immunity developed well in the mice bearing such metastases, but did not inhibit metastatic growth. Experiments using 125I-labelled SRBC or tumour cells revealed that such cells egressed rapidly from the BCG-primed site. When the tumour was inoculated into the contralateral foot to the BCG-primed site, the incidence and the number of metastases was reduced. Furthermore, BCG infection induced an increase of platelet count. I.v. injection of this tumour induced marked thrombocytopenia in normal mice. Administration of pentoxifylline, a methylxanthine derivative before tumour challenge reduced such metastases. These findings suggest that the changes in peripheral blood, such as increased platelet count and increased release of tumour cells from the injection site, facilitated distant metastasis in BCG-pretreated mice.     

Inhibitory effects of candesartan on responses to angiotensin peptides in the hindquarters vascular bed of the cat

Lactobacillus casei strain Shirota (LcS) has been shown to have potent anti-tumour and anti-metastatic effects on transplantable tumour cells and to suppress chemically-induced carcinogenesis in rodents. In particular, intrapleural (i.pl.) administration of LcS into tumour-bearing mice has been shown to effectively inhibit the growth of tumour cells in the thoracic cavity and to significantly prolong survival time. Also, i.pl. administration of LcS has been shown to induce the production of several cytokines, such as IFN-gamma, IL-1beta and TNF-alpha, in the thoracic cavity of mice, resulting in the inhibition of tumour growth and increased survival. On the other hand, oral administration of LcS has been shown to inhibit the growth of implantable tumour cells in rodents, and to restore the decreased mitogenic response of tumour-bearing mice. Administration of LcS has also been shown to inhibit chemically-induced bladder cancer in rodents. These findings suggest that treatment with LcS has the potential to ameliorate or prevent a variety of diseases through modulation of the host's immune system, specifically cellular immune responses.     

Effect of nucleic acid reactive antibodies on tumor cells grown in vivo

Nucleic acid reactive antibodies have been reported to inhibit various nucleic acid mediated functions in cell free systems. These antibodies were also shown to inhibit the growth of transformed cells in culture due to the high rate of endocytosis in transformed cells as compared to normal cells. In this report, we have tested the possibility of nucleic acid reactive antibodies inhibiting the growth of tumor cells in vivo. The life span of mice bearing Dalton's lymphoma ascites tumor cells was increased, when they were immunized with conjugates of guanosine-BSA, GMP-BSA and tRNA-MBSA complex before transplanting the tumor cells. A similar effect was also observed when mice were injected intraperitoneally with antibodies to guanosine or GMP along with the tumor cells. The specificity was ascertained, as immunization with non-specific antigens did not show any significant effect on tumor bearing mice. The results shows that nucleic acid reactive antibodies inhibit the growth of tumor cells in vivo.     

Creatine and cyclocreatine treatment of human colon adenocarcinoma xenografts: 31P and 1H magnetic resonance spectroscopic studies

Creatine (Cr) and cyclocreatine (cyCr) have been shown to inhibit the growth of a variety of human and murine tumours. The purpose of this study was to evaluate the anti-tumour effect of these molecules in relation to drug accumulation, energy metabolism, tumour water accumulation and toxicity. Nude mice carrying a human colon adenocarcinoma (LS174T) with a creatine kinase (CK) activity of 2.12 units mg(-1) protein were fed Cr (2.5% or 5%) or cyCr (0.025%, 0.1% or 0.5%) for 2 weeks and compared with controls fed standard diet. Cr concentrations of 2.5% and 5% significantly inhibited tumour growth, as did 0.1% and 0.5% cyCr. In vivo 31P magnetic resonance spectroscopy (MRS) after 2 weeks of treatment showed an increase in [phosphocreatine (PCr)+phosphocyclocreatine (PcyCr)]/nucleoside triphosphate (NTP) with increasing concentrations of dietary Cr and cyCr, without changes in absolute NTP contents. The antiproliferative effect of the substrates of CK was not related to energy deficiency but was associated with acidosis. Intratumoral substrate concentrations (measured by 1H-MRS) of 4.8 micromol g(-1) wet weight Cr (mice fed 2.5% Cr) and 6.2 micromol g(-1) cyCr (mice fed 0.1% cyCr) induced a similar decrease in growth rate, indicating that both substrates were equally potent in tumour growth inhibition. The best correlant of growth inhibition was the total Cr or (cyCr+Cr) concentrations in the tissue. In vivo, these agents did not induce excessive water accumulation and had no systemic effects on the mice (weight loss, hypoglycaemia) that may have caused growth inhibition.     

The effect of octreotide on wound healing: an in vitro and in vivo experimental study

OBJECTIVE: To investigate the effect of octreotide on wound healing. DESIGN: Experimental studies in vitro and in rats. SETTING: Teaching hospital, Israel. MATERIAL: Cultured human diploid fetal fibroblasts, and 36 male Wistar rats. INTERVENTIONS: Octreotide was added to cultures of fibroblasts in doses of 2, 10, 30, 60 and 120 ng/ml and fibroblasts were counted after 2, 4, and 6 days. Intestinal anastomoses were made in 36 rats. Rats in the octreotide group (n = 18) were given subcutaneous injections of 0.25 microg/kg twice daily and 6 rats were killed at 3, 7, and 14 days. The control group were given injections of saline. Anastomotic bursting pressures and hydroxyproline content were measured at each of the three times. MAIN OUTCOME MEASURES: Fibroblast counts, anastomotic bursting pressures, and hydroxyproline concentrations. RESULTS: Octreotide did not inhibit fibroblast proliferation in any of the doses at any of the time periods. The anastomotic bursting pressure was slightly higher in the octreotide group at each of the time points, but not significantly so, and there was no difference in hydroxyproline content between the octreotide and control groups. Octreotide did not inhibit wound healing either in vitro or in vivo.     

Some pharmacologic and toxicologic studies on Rhazya stricta decne in rats, mice and rabbits

1. This work examines some in vivo and in vitro pharmacologic and toxicologic effects of extracts of Rhazya stricta, a medicinal plant in the United Arab Emirates. 2. R. stricta extracts at doses of 0.1-10 mg reduced the mean arterial blood pressure (MBP) of anesthetized rats in a dose-dependent manner. The depressor effect was partially sensitive to atropine (5 microM). Although the MBP was reduced by 50% by both doses of extracts, the normal electrocardiogram pattern and the heart rate remained unaltered. 3. Acute treatment of rats with the lyophilized extract at doses of 4 g/kg produced a significant rise in insulin concentration. In streptozotocin-diabetic rats loaded orally with glucose (1 g/kg), R. stricta at doses of 8 g/kg produced significant decreases in plasma glucose concentration at 0.5 and 1 h after treatment. 4. Chronic treatment of rats and mice for 28 days with the lyophilized extract of R. stricta did not affect the plasma glucose or insulin concentration or any of the hematological or biochemical indices measured. 5. The extracts of R. stricta (0.5-4 g/kg) dose-dependently decreased the gastrointestinal transit time in mice by 4-50%. 6. The butanolic extract of R. stricta (1 and 2 g/kg) significantly reduced the carrageenan-induced increase in raw paw edema 3 and 4 h after the extract administration. 7. The rectal temperatures of normothermic and pyrexic rats were reduced significantly 0.5 and 1 h after administration of butanolic R. stricta at doses of 1 and 2 g/kg. 8. The butanolic extract of R. stricta at doses of 1 and 2 g/kg significantly increased the reaction time on the hot plate 30 and 60 min after administration to rats. 9. At concentration < 0.05 mg/ml (bath concentration), lyophilized water and butanol extracts of R. stricta potentiated the twitch responses induced by indirect electrical stimulation in the rat phrenic nerve diaphragm preparation. The responses were inhibited by concentrations > 0.05 mg/ml. Neostigmine (2 x 10(-4)M) did not alter these effects of the extracts. 10. R. stricta extracts dose-dependently decreased the force of contraction and heart rate of the isolated rabbit heart. Atropine (1 x 10(-5)M) had no effect on the inhibitory activity of these extracts. The lyophilized water extract (> 10 mg) and butanol extract (> 5 mg) produced irreversible inhibition and disturbances in the force of contraction and heart rate.     

NAT2, GSTM-1, cigarette smoking, and risk of colon cancer

There is overwhelming evidence to indicate that free radicals cause oxidative damage to lipids, proteins and nucleic acids and are involved in the pathogenesis of several degenerative diseases. Therefore, antioxidants, which can neutralize free radicals, may be of central importance in the prevention of these disease states. The protection that fruits and vegetables provide against disease has been attributed to the various antioxidants contained in them. Recently, an anti-inflammatory and analgesic activity of a water-soluble fraction from shark cartilage has been described. Using electrophoretical assays, bacteria survival and transformation and the Salmonella/mammalian-microsome assay, we investigated the putative role of shark cartilage-containing preparation in protecting cells against reactive oxygen species induced DNA damage and mutagenesis. If antimutagens are to have any impact on human disease, it is essential that they are specifically directed against the most common mutagens in daily life. Our data suggest that shark cartilage-containing preparation can play a scavenger role for reactive oxygen species and protects cells against inactivation and mutagenesis.     

Manipulation of the immune response of mice against neu/HER2-expressing tumours

The rat oncogene neu, and its human homologue HER2, can both cause cell transformation in vitro and tumour formation in vivo, albeit by different mechanisms. 3T3 cells (B104.1.1) transfected with mutated neu (neu) grow as solid tumours in Swiss mice. The purpose of this study was to determine whether immunization with extracts of different 3T3 lines expressing these oncoproteins would lead to a cross-reactive response, and whether this response could alter B104. 1.1 tumour growth. Both humoral and cellular cross-reactive responses were observed, which were capable of inhibiting tumour growth in vivo. This cross-reactive response may be relevant to the immunotherapy of HER2-expressing tumours in humans.     

FGF signal transduction in PC12 cells: comparison of the responses induced by endogenous and chimeric receptors

Loss of articular cartilage, which is the most important pathological lesion occurring in osteoarthritis, has been shown to be enzymatically mediated. The matrix metalloproteinases (MMPs) are a group of enzymes which have been implicated in this degradation of articular cartilage matrix. The use of pharmacological agents to inhibit this catabolic process in the joint is a potential route for therapeutic intervention. The gelatinase MMPs, MMPs-2 and 9, were purified by affinity chromatography from equine cell cultures. The ability of phenylbutazone, flunixin, betamethasone, dexamethasone, methylprednisolone acetate (MPA), hyaluronan, pentosan polysulphate and polysulphated glycosaminoglycan (PSGAG) to inhibit equine MMPs-2 and 9 were assessed by two degradation assays. Whilst some agents did have direct effects on MMP activity, these effects were only obtained at concentrations which were unlikely to be achieved for any length of time in vivo. It is improbable that any pharmacological agent, currently used in the horse, has a significant effect on gelatinase MMP activity.     

Evaluation of carborane-containing porphyrins as tumour targeting agents for boron neutron capture therapy

A number of carborane-containing porphyrins were administered to mice bearing subcutaneously transplanted mammary carcinomas. Administration was via serial intraperitoneal (i.p.) injections to assess their relative toxicities and tumour affinities. Three analogues of the natural porphyrin heme and four tetraphenylporphyrins (TPPs) were given at total doses of 78-245 micrograms g-1 body weight. The water-insoluble TPPs were less toxic to mice, and delivered greater amounts of boron to tumour than did the water-soluble TPPS and the heme analogues. One such compound, NiTCP-H, delivered more than 100 micrograms B g-1 to tumour tissue with a tumour:blood boron concentration ratio greater than 500:1 and a tumour: brain boron concentration ratio greater than 50:1, 4 days after the last of six i.p. injections given over 2 days. Another TPP analogue, NiTCP, delivered approximately 50 micrograms B g-1 to tumour with similar boron concentrations in normal tissues. Neither compound was toxic to mice at total doses of approximately 200 micrograms g-1 body weight. In contrast, the heme analogues were toxic and, with the exception of VCDP, delivered less boron to tumour than NiTCP and NiTCP-H. The two porphyrins with the greatest potential for application to boron neutron capture therapy (BNCT), NiTCP and NiTCP-H, yielded higher tumour:blood and tumour:brain boron concentration ratios in mice than could be achieved with p-boronophenylalanine (BPA) and sodium mercaptoundecahydrododecaborate (BSH), the compounds which are currently being used in clinical trials of BNCT in the treatment of glioblastoma. The boron delivered by each of the porphyrins tested remained in tumour tissue longer than did boron delivered by either BPA or BSH. The copper and nickel chelates of these porphyrins behave identically in vivo. The former offer the potential for imaging by 67Cu-mediated single photon emission computed tomography (SPECT) to aid BNCT treatment planning.     

Differential inhibition of fluid accumulation and tumor growth in two mouse ascites tumors by an antivascular endothelial growth factor/permeability factor neutralizing antibody

In the accompanying paper (Luo et al., Cancer Res., 58: 2652-2660, 1998), we demonstrated that vascular endothelial growth factor (VEGF), also designated vascular permeability factor (VPF), significantly accumulated in all mouse malignant ascites tested, suggesting its fundamental role in ascites tumors. Removal of VEGF may inhibit the development of ascites tumors. In this study, using a goat antimouse VEGF-neutralizing antibody, we tested this hypothesis with two well-defined syngeneic mouse ascites tumors: MM2 breast adenocarcinoma and OG/Gardner lymphoma 6C3HED (expressing moderate and low levels of VEGF, respectively). This antibody significantly inhibited MM2 and OG cell-free ascites fluid-induced hyperpermeability of mouse peritoneal microvessels and in vitro endothelial cell growth. Mice bearing tumors were administered i.p. daily with the antibody or normal goat IgG as controls for 8 days, at doses of 20-fold (for MM2-bearing mice) or 40-fold (for OG-bearing mice) the estimated amounts of VEGF that kinetically accumulated in the ascites fluid after the tumor inoculation. The average volume of ascites fluid, number of tumor cells and leaked RBCs, and the peritoneal microvessel permeability in MM2-bearing mice that received the antibody treatment were significantly lower than those in the matched controls (P < 0.01). Unexpectedly, OG-bearing mice did not show satisfactory response to the anti-VEGF treatment. This discrepancy was not likely due to inadequate doses or different host immune responses, but it was quite possibly to the different characteristics of MM2 carcinoma and OG lymphoma tumors, the latter being strongly invasive, and/or the existence of an inflammatory mediator(s), such as bradykinin or cytokine(s) other than VEGF. In summary, our results directly demonstrated, for the first time, differential roles for VEGF in ascites tumors in vivo and suggest the potential of VEGF inhibition as a specific therapy for ascites tumors of carcinoma origin, which are the major cause of the malignant ascites in adult humans.     

1H MRS markers of tumour growth in intrasplenic tumours and liver metastasis induced by injection of HT-29 cells in nude mice spleen

We have characterized, by in vitro magnetic resonance spectroscopy (MRS), the metabolite pattern of perchloric acid (PCA) extracts of intrasplenic tumours and hepatic metastasis, produced by intra-spleen injection of the human colorectal carcinoma cell line HT-29 and its metastatic variant HT-29 MMM into nude mice. Our aim was to gain further understanding of colorectal tumour metabolism as a basis for future in vivo studies of human colon cancer by 1H MRS. Metabolite PCA extract analysis showed a good reproduction of the spectral pattern observed in human primary colon tumours, while they were very different from the spectral pattern of the host tissues (spleen and liver). The main differences between host and tumour tissues involved taurine, phosphocholine (PC), phosphoethanolamine (PE), creatine, glycogen and glucose. Creatine is the most promising marker to follow tumour growth because of its practical absence in the nude mice host tissues. Detection of variable levels of this compound and of taurine in hepatic foci in man, are suggested as possible diagnostic markers. No correlation could be found between spectral pattern differences and the different ability to metastasize of the two HT-29 cell lines used. Furthermore, indirect evidence for a functional link between taurine and myo-inositol in colon tumour cells is presented. In summary, our data suggest that the nude mice model may be a suitable system for the MRS study of the changes taking place in host tissues upon tumour progression.     

The intersex difference in the production of alloantibodies and growth of tumour allografts in mice

The comparisons of immune responses to tumour allografts in male and female mice showed the following intersex differences: 1. Production of humoral antibodies is, in general, higher in females; since, however, the time course of titres differs for haemagglutinating and cytotoxic antibodies as well as with the sex, the titre of the same antibody may be higher in males at certain time intervals when the peak is delayed and the production in females is already declining. 2. In recipients specifically presensitized with spleen extract as well as in controls, the growth of allogeneic tumour (SaI) exhibits quantitative intersex differences being reduced in females. In the initial phase of growth, following the spleen extract, the facilitation (enhancement) of the tumour allograft is apparent olny in males, whereas in females it is delayed. 3. The facilitating effect of antibodies passively transferred from males or females corresponds mainly to the sex and strain of the recipient, only rarely to the sex of the producer. 4. In CBA strain mice, females are more resistant to the growth of SaI and also mortality from the primary tumour is lower in females; in contrast, the frequency of metastases in females is higher. 5. In the IC strain, where the sex effect on tumour growth is particularly pronounced, the consequences of castration were concordant with the inferred hormonal control of the allograft response: in males, the relatively high percentage of lethal spontaneous takes (60%) was reduced following castration (to 13%), whereas the opposite trend was observed in females, namely an increase from 0% in sham-operated controls to 23% in the castrated group.     

Effect of treatment with 13-cis-retinoic acid and ara-C on the hematopoietic stem cells of patients with myelodysplastic syndromes

PURPOSE: To assess the "in vivo" effect of 13-cis-retinoic acid and low dose Ara-C in MDS as well as to establish "in vitro" advantage of retinoid dose-related growth pattern on bone marrow cultures as defined by culture timing and CFU-GM proliferative response. PATIENTS AND METHODS: We evaluated 28 patients diagnosed of MDS according to FAB classification, of whom 4 cases had RA, 8 cases SRA, 14 cases RAEB and 2 cases RAEB-T. Patients who had RA and SRA were treated with oral 13-cis-retinoic acid at doses of 20-40 mg daily for 4 months and those cases with RAEB and RAEB-T had subcutaneous Ara-C at doses of 3 mg/m2 twice a day for 21 days. The "in vivo" and "in vitro" effect of retinoic acid on the haemopoietic differentiation was evaluated by the growth CFU-GM in semisolid cell culture methods. RESULTS: Increasing in vitro concentrations of 13-cis retinoic acid did not enhance the growth of myelodysplastic progenitors. Nevertheless, our study did not find any beneficial therapeutic effect of retinoic compounds in MDS patients. In this study, low-dose Ara-C (3 mg/m2) showed similar effects when compared with higher doses reported by others. Furthermore, in terms of CFU-GM proliferation the concentration of colonies before and after treatment were fairly similar in all but two patients. CONCLUSIONS: The results drawn from our study demonstrated that there is no beneficial advantage of 13-cis-retinoic acid as a differentiation inducing agent on myelodysplastic patients. In contrast, lower doses of Ara-C showed similar effects on haemopoiesis of MDS patients than standard doses of 10-20 mg/m2 but with less side effects.     

Correction of left handedness worsened mirror writing in a girl with spastic diplegia

We investigated the antitumor effect of vitamin A(VA) using the double grafted tumor technique to examine whether VA administered into a primary tumor (intralesionally or i.l.) accelerates antitumor immune reactions so that growth of the secondary tumor may be more effectively inhibited than by other systemic administration routes. In the double grafted tumor system, where BALB/c mice were inoculated with MethA fibrosarcoma cells into the right inguinal region (1 x 10[6] cells) on day 0 and later into the left (3 x 10[6] cells) on day 10, the injection of VA at a dose of 1000 IU/mouse i.l., s.c., i.p., and i.v. on days 3 through 7 inhibited the growth of the secondary tumor to the same extent, while VA at the i.l. dose of 100 IU/mouse into the primary tumor inhibited more effectively than by any other administration route. VA did not inhibit the secondary MethA growth in BALB/c (nu/nu) mice. The spleen cells taken from VA-treated tumor-bearing mice prevented the growth of MethA tumors in naive BALB/c mice when given as a mixture with the MethA inoculum (the Winn assay). The delayed-type hypersensitivity (DTH) response to methylated bovine serum albumin (MBSA) antigen was augmented when VA (1000 IU) was injected at the site of the antigen injection. These results suggest that the direct interaction of VA with the tumor cells may be necessary for the tumor immunity-potentiating effect of VA, and that T-lymphocyte-mediated tumor immunity is involved in the anti-tumor effect of VA. The antitumor mechanism of VA seems to involve retinoid receptors, because the benzoic acid derivative Am80, which has been reported to exert retinoidal activity by binding to specific retinoid receptors, also showed activity.     

Toxicity, antitumor and chemosensitizing effects of 3-chloroprocainamide

3-Chloroprocainamide (3-CPA), an analog of metoclopramide (MCA), dose-dependently inhibited tumor growth in scid mice xenografted with a human brain astrocytoma (T24) when given intramuscularly to mice every third day for 14-20 days. 3-CPA was shown to have the same efficacy on tumor growth inhibition as neutral metoclopramide (neutral MCA) at the doses of 10-40 mg/kg when evaluated by tumor doubling time, tumor growth time for tumor volumes to reach 1000 mm3 and area under growth curve. 3-CPA at the dose of 3 x 40 mg/kg was also shown to enhance the cytotoxicity induced by a single dose of cisplatin at 7.5 mg/kg. A dose of < or = 160 mg/kg of 3-CPA did not show any notable extrapyramidal symptoms which was observed for neutral MCA treated mice at the dose of 20 mg/kg. The lethal response dose of 3-CPA for scid mice was 320 mg/kg which is 4 times higher than that determined for neutral MCA (80 mg/kg). These results support 3-CPA as a good candidate drug representing a new generation of benzamides for further clinical development as a cancer therapy drug.     

Combined effects of angiostatin and ionizing radiation in antitumour therapy

Angiogenesis, the formation of new capillaries from pre-existing vessels, is essential for tumour progression. Angiostatin, a proteolytic fragment of plasminogen that was first isolated from the serum and urine of tumour-bearing mice, inhibits angiogenesis and thereby growth of primary and metastatic tumours. Radiotherapy is important in the treatment of many human cancers, but is often unsuccessful because of tumour cell radiation resistance. Here we combine radiation with angiostatin to target tumour vasculature that is genetically stable and therefore less likely to develop resistance. The results show an antitumour interaction between ionizing radiation and angiostatin for four distinct tumour types, at doses of radiation that are used in radiotherapy. The combination produced no increase in toxicity towards normal tissue. In vitro studies show that radiation and angiostatin have combined cytotoxic effects on endothelial cells, but not tumour cells. In vivo studies show that these agents, in combination, target the tumour vasculature. Our results provide support for combining ionizing radiation with angiostatin to improve tumour eradication without increasing deleterious effects.     

Kinetics of cell proliferation and cell loss in the peripheral and central parts of Walker tumours growing in rats and nude mice

In previous experiments it was shown that, in the submucosal part of Walker tumours transplanted to the gastric wall of rats, a lower rate of cell proliferation was seen in the peripheral zone, defined as the outer 100-120 mu of the tumours, than in the main tumour mass. The purpose of the present experiments was to investigate whether such differences are independent of the location of the Walker tumour, or were caused by local factors specific for the gastric mucosa, and whether specific cellular immunity cell proliferation at the periphery of a transplanted tumour. Cells from Walker 256 tumour were injected into the subcutaneous space in rats and in mutant nude mice, which lack T lymphocytes. In one series, the rats and mice were injected with 3H-TDR at different time intervals before sacrifice. In a second series vinblastine sulfate was injected 3 hours before sacrifice. Although all the animals were given the same tumour dose, the tumours in mice increased in size more slowly than those in rats. In the first-mentioned series, the mitotic counts, the labelled cells and the percentage labelled mitoses (PLM) in the main tumour mass and at the tumour perphery were counted. In the second series the mitotic rate in the same two regions was determined. A significantly lower rate of cell proliferation was demonstrated at the periphery compared to the main tumour mass in both rats and mice. Differences between the PLM curves in the two regions were also found. Possible explanations of these findings are discussed. It is concluded that the described growth pattern is probably a general characteristic of the Walker tumour, and that the low rate of proliferation at the periphery is not caused by specific immunological mechanisms mediated through T lymphocytes. If the growth rates were calculated on the assumtion that the actual tumour growth followed a Gompertz function, then the rate of cell loss in the tumour in mice was higher than that in the tumour in rats.     

Progressive polyarthritis induced in BALB/c mice by aggrecan from normal and osteoarthritic human cartilage

OBJECTIVE: To find an "unlimited" source of antigenic material (aggrecan) for arthritis induction in BALB/c mice; to analyze the specificities of immune reactions to aggrecan and type II collagen in 2 arthritis-susceptible murine strains, BALB/c mice for proteoglycan (aggrecan)-induced arthritis and DBA/1j mice for collagen-induced arthritis; to compare the histopathologic features of arthritis induced by purified aggrecans or total extracts of osteoarthritic (OA) cartilage; and to determine arthritis susceptibility in various BALB/c colonies. METHODS: Aggrecans from total extracts of human fetal, normal adult, OA, and rheumatoid cartilage samples and from osteophytes were isolated, purified by gradient centrifugation, deglycosylated, characterized, and tested for arthritis induction. Purified type II collagen and salt-soluble collagens from OA cartilage were denatured, stromelysin treated, and used for immunization and arthritis induction in arthritis-susceptible (DBA/1j and BALB/c) murine strains. RESULTS: Chondrocytes from OA cartilage synthesize predominantly fetal-type aggrecan, which is the most efficient antigenic material for arthritis induction in BALB/c mice. The critical autoimmune/arthritogenic T cell epitopes of aggrecan are located in the G1 domain. Although most of the aggrecan molecules are heavily degraded and lost from OA cartilage, the G1 domain-containing fragments accumulate in OA cartilage. The amount of G1-containing fragments is approximately twice as much in OA than in normal adult articular cartilage, and the arthritogenic epitope(s) remains intact in G1-containing fragments retained in cartilage. Thus, total extracts of OA cartilage (without additional purification), if deglycosylated appropriately, can be used as arthritogenic material in BALB/c mice. CONCLUSION: Predominantly G1 domain-containing fragments of aggrecan accumulate in OA cartilage, and these are the fragments which induce arthritis in BALB/c mice. Arthritis induction is highly specific for aggrecan epitopes and dictated by the genetic background of the BALB/c strain.     

Organogenesis and angiogenin

Our search for an angiogenesis-inducing factor in culture medium conditioned by human colon adenocarcinoma cells (HT-29) was inspired by the 'organizer' hypothesis originally postulated by Spemann. It led us to the isolation of angiogenin, a 14 kD protein homologous to pancreatic ribonuclease and one of the most potent stimulators of blood vessel formation known. This review summarizes the properties of angiogenin, its enzymatic and three-dimensional relationship to ribonuclease A (RNase A), those aspects of its structure that are critical for its biological function, and the therapeutic potential of angiogenin inhibition. Despite having the same arrangement of catalytic residues as RNase A, angiogenin has very low enzymatic activity. It lacks one of the four disulphide loops of RNase A; instead, the corresponding residues form part of a cell binding region. Both the catalytic activity and cell binding site are essential for angiogenesis. Angiogenin binds to cell-surface actin in confluent endothelial cells and to an as yet uncharacterized receptor on proliferating cells. Internalization and translocation to the nucleolus are also required for activity. Inhibitors of angiogenin can block angiogenesis in vitro and prevent tumour growth in vivo. Thus, a noncytotoxic neutralizing monoclonal antibody prevents the establishment of HT-29 human tumour xenografts in up to 65% of treated athymic mice. In those tumours that develop, the number of vascular elements is reduced. Actin also prevents the establishment of tumours while exhibiting no toxic effects at daily doses > 50 times the molar amount of circulating mouse angiogenin. These antagonists also inhibit the appearance of tumours derived from two other human tumour cell lines. Inhibition of the action of angiogenin may prove to be an effective therapeutic approach for the treatment of malignant disease.