The angiogenesis inhibitor vasostatin does not impair wound healing at tumor-inhibiting doses.

Inhibition of tumor angiogenesis represents a promising new approach for the treatment of human cancers. It has remained unclear, however, whether inhibition of tumor angiogenesis may also result in impaired wound healing, a process thought to be angiogenesis dependent. To determine the effects of the angiogenesis inhibitor vasostatin, a 180 amino acid calreticulin fragment, on wound healing at tumor inhibiting doses, full-thickness wounds were generated on the back of nude mice that were also injected intradermally with CA46 Burkitt lymphoma cells. Mice were treated with daily injections of vasostatin or vehicle control at a site between the wounds and the transplanted tumor cells over 14 d. Vasostatin potently inhibited tumor growth and significantly reduced tumor angiogenesis, as measured by computer-assisted image analysis of CD31-stained tumor sections. Moreover, vasostatin treatment resulted in an increased fraction of mature tumor-associated blood vessels. In contrast, no impairment of wound healing was observed in vasostatin-treated mice, despite a significantly reduced vascularity of the wound granulation tissue. Our results reveal a different sensitivity of malignant tumor growth and physiologic wound healing to inhibition of angiogenesis, and they suggest that therapeutic inhibition of tumor angiogenesis may be achieved without impairment of tissue repair.

Use of cough swabs in a cystic fibrosis clinic.

We audited prospectively 322 cough swabs taken from cystic fibrosis children and compared cough swabs with concomitant sputum samples in 30 expectorating patients. A positive cough swab is a strong predictor of sputum culture. However, a negative cough swab does not rule out infection. Persistent symptoms should be further investigated.

Effect of intravenous antibiotics on exercise tolerance (3-min step test)) in cystic fibrosis.

Most children with cystic fibrosis (CF) feel better and display more energy after a course of intravenous antibiotics (IVABs), but this is not always reflected by a satisfactory improvement in lung function. We assessed the change in exercise tolerance after treatment with IVABs using the 3-min step test, and compared it with changes in spirometric lung function and arterial oxygen saturation (SaO(2)). Thirty-six children (mean age, 13.8 years) were enrolled from two tertiary CF centers during an inpatient stay for IVABs. After 10-14 days of treatment, there was a significant improvement in median FEV(1) from 43% to 57% of predicted values (P < 0.0001), and median FVC from 66% to 73% of predicted values (P < 0.0001), while median SaO(2) significantly increased from 95% to 96.5% (P < 0.05). This was accompanied by a reduction in resting heart rate (median 118 bpm to 109 bpm, P < 0.005) and subjective breathlessness at rest (median visual analogue score 2.2 to 0.8, P < 0.005). All outcomes of exercise tolerance were improved after IVABs. There was a reduction in maximum heart rate (median 156 bpm to 150 bpm, P < 0.05) and an increase in minimum SaO(2) (median 93.5% to 94.5%, P = 0.08) measured during the step test. There was also a reduction in subjective breathlessness (median visual analogue score of 5.5 to 4.2, P < 0.005) and objective breathlessness (median 15-count score of 3 to 2, P < 0.0001) measured immediately after the step test. Exercise testing was a useful outcome measure for monitoring effectiveness of inpatient therapy, and complemented spirometry and SaO(2) monitoring. The simple ward-based 3-min step test was found to be a particularly suitable method for measuring changes in exercise tolerance in children with CF.

Measurement of polyclonal immunoglobulin synthesis using the reverse plaque technique.

This unit describes the reverse hemolytic plaque assay, an effective method for measuring the number of immunoglobulin (Ig)-secreting cells present in a cell population at any particular time. Cell populations that can be assayed using the technique include peripheral blood mononuclear cells or cells from tissues such as the tonsils. The basic protocol is divided into three stages. First, protein A-sensitized sheep red blood cells (SRBC), guinea pig complement, and anti-Ig antibody are prepared. Test samples are then combined and incubated with the SRBC, complement, and antibody in appropriate chambers. Finally, the resulting plaques are scored. A support protocol describes the preparation of plaquing chambers.

Effective targeting of tumor vasculature by the angiogenesis inhibitors vasostatin and interleukin-12.

Solid tumors are dependent on preexisting vasculature and neovascularization for their growth. Successful cancer therapies targeting the tumor vasculature would be expected to block the existing tumor blood supply and to prevent tumor neovascularization. We tested the antitumor activity of experimental therapy with 2 distinct antiangiogenic drugs. Vasostatin inhibits endothelial cell growth and neovascularization, and interleukin-12 (IL-12) targets the tumor vasculature acting through interferon-gamma (IFN-gamma) and the downstream chemokines interferon-inducible protein-10 (IP-10) and monokine induced by IFN-gamma. Individually, vasostatin and IL-12 produced distinct efficacy profiles in trials aimed at reducing tumor growth in athymic mice. In combination, these inhibitors halted the growth of human Burkitt lymphoma, colon carcinoma, and ovarian carcinoma. Thus, cancer therapy that combines distinct inhibitors of angiogenesis is a novel, effective strategy for the experimental treatment of cancer. (Blood. 2000;96:1900-1905)

Calreticulin and calreticulin fragments are endothelial cell inhibitors that suppress tumor growth.

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.

Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth.

An endothelial cell inhibitor was purified from supernatant of an Epstein-Barr virus-immortalized cell line and identified as fragments of calreticulin. The purified recombinant NH2-terminal domain of calreticulin (amino acids 1-180) inhibited the proliferation of endothelial cells, but not cells of other lineages, and suppressed angiogenesis in vivo. We have named this NH2-terminal domain of calreticulin vasostatin. When inoculated into athymic mice, vasostatin significantly reduced growth of human Burkitt lymphoma and human colon carcinoma. Compared with other inhibitors of angiogenesis, vasostatin is a small, soluble, and stable molecule that is easy to produce and deliver. As an angiogenesis inhibitor that specifically targets proliferating endothelial cells, vasostatin has a unique potential for cancer treatment.

Holding the baby: head downwards positioning for physiotherapy does not cause gastro-oesophageal reflux.

The head-downwards tipped position for physiotherapy has been claimed to exacerbate gastro-oesophageal reflux (GOR) in infants with cystic fibrosis (CF). This was investigated using lower oesophageal pH monitoring during physiotherapy. Twenty-one infants (age range 1-27 months) with respiratory disorders (CF=11), undergoing lower oesophageal pH monitoring were recruited. Subjects received two physiotherapy episodes in random order, A/B or B/A, 12 h apart. A began the gravity-assisted positioning head downward tip for: right lower lobe, middle lobe, left lower lobe and lingula; then supine with no tip for anterior segments of the upper lobes followed by apical segments of upper lobes in a sitting position. B was in the reverse order. Intermittent chest clapping was carried out for 4 min in each position by a physiotherapist blinded to the pH data. During episode A, the median change in pH from baseline was -0.32 (range -2.07 to +1.0) in non-CF subjects (NS) and -0.52 (range -2.7 to +0.52) in CF subjects (p<0.02). During episode B, the median change in non-CF subjects was -0.1 (NS; range - 1.7 to -0.15) and in CF subjects was -0.05 (NS; range -0.67 to +0.5). There was no order effect for positioning. In the CF subjects the sitting position was twice as likely to have the lowest pH measurement during physiotherapy than the other positions (p<0.04). In conclusion, the head-downward tipped positioning for physiotherapy treatment neither induces nor aggravates gastro-oesophageal reflux. There is no justification for routinely changing the way in which infant physiotherapy is carried out.

Role of the p53 tumor suppressor gene in the tumorigenicity of Burkitt's lymphoma cells.

Burkitt's lymphoma (BL) cell lines carry a translocated c-myc gene and, in 60-80% of cases, exhibit mutations in the p53 tumor suppressor gene. We examined the potential role of the p53 gene in BL tumorigenicity using an in vitro assay that measures p53-dependent cell cycle arrest in the G1 phase of the cell cycle and an in vivo athymic murine model that detects differences in the tumorigenicity of BL cell lines. A highly significant inverse correlation was found between the ability of BL cells to arrest in G1 after irradiation and their tumorigenicity in athymic mice, consistent with the notion that loss of p53 function is associated with increased tumorigenicity. Inactivation of wild-type (wt) p53 function by expression of the human papillomavirus E6 protein in the AG876V BL cell line, which carries both wt and mutant p53 proteins, rendered the cell line significantly more tumorigenic in athymic mice. Transfection of the wt p53 gene into the p53 mutant and highly tumorigenic BL-41 cell line caused it to acquire wt p53 function and rendered it less tumorigenic in mice. In addition to confirming a role for the loss of p53 function in tumor progression, the data demonstrate that wt p53 protein can reduce BL tumorigenicity in vivo.

Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo.

Human Burkitt lymphoma cell lines give rise to progressively growing subcutaneous tumors in athymic mice. These tumors are induced to regress by inoculation of Epstein-Barr virus-immortalized normal human lymphocytes. In the present study, analysis of profiles of murine cytokine/chemokine gene expression in Burkitt tumor tissues excised from the nude mice showed that expression of the murine alpha-chemokine interferon-inducible protein-10 (IP-10) was higher in the regressing than in the progressive Burkitt tumors. We tested the effects of IP-10 on Burkitt tumor growth in nude mice. Inoculation of established Burkitt tumors either with crude preparations of murine IP-10 or with purified human IP-10 caused visible tumor necrosis in a proportion of the animals, although no complete tumor regressions were observed. Constitutive expression of murine IP-10 in Burkitt cells reduced their ability to grow as subcutaneous tumors, and caused visible tumor necrosis in a proportion of the animals. Histologically, IP-10-treated and IP-10-expressing Burkitt tumors had widespread evidence of tumor tissue necrosis and of capillary damage, including intimal thickening and vascular thrombosis. Thus, IP-10 is an antitumor agent that promotes damage in established tumor vasculature and causes tissue necrosis in human Burkitt lymphomas established subcutaneously in athymic mice.

Regression of experimental Burkitt's lymphoma induced by Epstein-Barr virus-immortalized human B cells.

Epstein-Barr virus (EBV)-immortalized human B cells survive only transiently when injected subcutaneously into athymic mice, whereas Burkitt's lymphoma cells give rise to progressively growing subcutaneous tumors. In this study, we tested whether these Burkitt's tumors could be induced to regress via a bystander effect induced by EBV-immortalized B cells. Simultaneous inoculation of EBV-immortalized B cells and Burkitt's lymphoma cells in the same subcutaneous site resulted in tumors that regressed with necrosis and scarring. Similarly, simultaneous inoculation of EBV-immortalized B cells and Burkitt's lymphoma cells in separate subcutaneous sites resulted in regression of a proportion of the Burkitt's tumors. Furthermore, most of the established human Burkitt's tumors regressed with necrosis and scarring after intratumor inoculations with EBV-immortalized B cells. The EBV-immortalized B cells continued to exert this antitumor effect even when killed with irradiation. The experimental approach to Burkitt's lymphoma treatment described here exploits the ability of athymic mice to reject EBV-immortalized B cells to target an effective antitumor response to malignant cells normally incapable of eliciting it.

A mechanism of T cell regulation of Epstein-Barr virus latency.

The tumorigenic potential of B lymphocytes latently infected with EBV is effectively controlled by T cell immunity. The mechanisms of this T cell regulation, however, are incompletely understood. In this study, T lymphocytes were found to proliferate in response to serum-free supernatants of EBV-immortalized cells and to deplete them of growth factors required by the immortalized B cells for autocrine growth. Lactic acid was reported to account for approximately 90% of the autocrine growth factor activity in serum-free supernatants of EBV-immortalized cell lines. Synthetic lactic acid was now found to promote growth in activated T cells. In addition, B cell suppression resulting from coculture of EBV-infected B cells with autologous T cells was reversed by the addition of supernatants from EBV-immortalized cell lines. Thus, T cell competition for growth factors produced and utilized by EBV-immortalized B cells for continuous proliferation may represent an important and novel regulatory mechanism for the maintenance of EBV latency in B lymphocytes.

The role of lactic acid in autocrine B-cell growth stimulation.

Growth and survival of Epstein-Barr virus (EBV)-immortalized B lymphocytes cultured at low cell densities require autocrine soluble factors. In this study, we have purified a low molecular weight autocrine soluble factor that promotes growth of EBV-immortalized B cells in serum-free conditions and identified it as lactic acid (LA). Synthetic LA stimulated growth in EBV-immortalized B cells at 1-10 mM, a concentration of LA measured in the culture supernatant of EBV-immortalized cell lines. LA alone was found to account for greater than 70% of the autocrine growth factor activity in serum-free supernatants of EBV-immortalized B cells. Aminooxyacetate, a glutamate-oxaloacetate transaminase inhibitor, specifically inhibited B-cell growth induced by LA, suggesting that this process requires mitochondrial-cytosol transfers. Thus, LA is an autocrine stimulatory molecule that in serum-free conditions is essential for the continuous proliferation of EBV-immortalized B cells. This represents an unexpected function for LA.

Identification of interleukin-6 as an autocrine growth factor for Epstein-Barr virus-immortalized B cells.

Autocrine growth factors are believed to be important for maintenance of an immortalized state by Epstein-Barr virus (EBV), because cell-free supernatants of EBV-immortalized cell lines promote the proliferation of autologous cells and permit their growth at low cell density. In this study, we provide evidence for the existence of two autocrine growth factor activities produced by EBV-immortalized lines distinguished by size and biological activities. Much of the autocrine growth factor activity in lymphoblastoid cell line supernatants resided in a low-molecular-weight (less than 5,000) fraction. However, up to 20 to 30% of the autocrine growth factor activity resided in the high-molecular-weight (greater than 5,000) fraction. While the nature of the low-molecular-weight growth factor activity remains undefined, the high-molecular-weight growth factor activity was identified as interleukin-6 (IL-6). Culture supernatants from six EBV-induced lymphoblastoid cell lines tested contained IL-6 activity, because they promoted proliferation in the IL-6-dependent hybridoma cell line B9. In addition, a rabbit antibody to human IL-6 neutralized the capacity of the high-molecular-weight (greater than 5,000) fraction of a lymphoblastoid cell line supernatant to promote growth both in autologous EBV-immortalized cells and in B9 cells. Similarly, this high-molecular-weight autocrine growth factor activity was neutralized by a monoclonal antibody to human IL-6. Furthermore, characteristic bands, attributable to IL-6, were visualized in supernatants of each of four EBV-induced lymphoblastoid cell lines after immunoprecipitation with a rabbit antiserum to human IL-6. Thus, in addition to its previously reported properties, IL-6 is an autocrine growth factor for EBV-immortalized B cells cultured under serum-free conditions.

Accessory function of interleukin-1 and interleukin-6: preferential costimulation of T4 positive lymphocytes.

Two monocyte-derived cytokines, interleukin-1 (IL-1) and interleukin-6 (IL-6), have been reported to costimulate monocyte-depleted T cell populations in the presence of mitogen, and this effect has been attributed to an accessory function of these molecules. We have now examined further the accessory function potential of IL-1 plus IL-6, and examined how these cytokines promote T cell growth with mitogen. Together, IL-1 and IL-6 additively and, to a small degree, synergistically promote the proliferation of highly purified human peripheral blood T cells with phytohemagglutinin (PHA). However, maximum costimulation by IL-1 plus IL-6 over a wide range of concentrations is significantly smaller than that induced by optimal numbers of monocytes. Also, in contrast to monocytes that costimulate equally effectively T4 positive and T8 positive cells, IL-1 plus IL-6 costimulate T4 positive lymphocytes in marked preference to T8 positive cells. IL-1 plus IL-6 induces IL-2 secretion in T cell cultures costimulated with PHA, and an antibody to the IL-2 receptor, anti-Tac, markedly inhibits PHA-activated T cells costimulated by IL-1 plus IL-6. In addition, IL-1 plus IL-6 enhances the expression of surface IL-2 receptors. Because the costimulatory effect of IL-1 plus IL-6 is quantitatively smaller than that of monocytes, and it is preferentially directed toward T4 positive as opposed to T8 positive T cells, IL-1 plus IL-6, together, appear to represent a selective set of monocyte-derived accessory signals.

Interferon-beta 2/interleukin 6 is a co-stimulant for human T lymphocytes.

IFN-beta 2/IL-6 promotes the proliferation of human peripheral blood T cells in the presence of either PHA or Con A. This effect is observed with highly purified T cells and is masked by the addition of as few as 2% monocytes, suggesting that accessory cells are not required and that IFN-beta 2/IL-6 acts directly on the T cells. Both T4-positive and T8-positive cells respond to IFN-beta 2/IL-6 plus PHA. Studies of the time requirements of IFN-beta 2/IL-6 and of PHA in the response of T cells show that optimal co-stimulation occurs when both IFN-beta 2/IL-6 and PHA are added together at the outset of culture, suggesting that IFN-beta 2/IL-6 acts predominantly on resting T cells. Unlike T cell proliferation induced by IL-2, T cell proliferation induced by IFN-beta 2/IL-6 is not blocked by antibodies to the IL-2 R, suggesting that IFN-beta 2/IL-6 does not act by stimulating IL-2 production. Thus, in addition to its previously reported properties, IFN-beta 2/IL-6 stimulates T cells in the presence of mitogen, and may prove to be of considerable importance in the physiologic activation of T cells.

Stimulation of EBV-activated human B cells by monocytes and monocyte products. Role of IFN-beta 2/B cell stimulatory factor 2/IL-6.

EBV infects human B lymphocytes and induces them to proliferate, to produce Ig, and to give rise to immortal cell lines. Although the mechanisms of B cell activation by EBV are largely unknown, the continuous proliferation of EBV-immortalized B cells is dependent, at least in part, upon autocrine growth factors produced by the same EBV-infected B cells. In the present studies we have examined the influence of monocytes on B cell activation by EBV and found that unlike peripheral blood T cells and B cells, monocytes enhance by as much as 30- to 50-fold virus-induced B cell proliferation and Ig production. Upon activation with LPS, monocytes secrete a growth factor activity that promotes both proliferation and Ig secretion in EBV-infected B cells and thus reproduces the effects of monocytes in these cultures. Unlike a number of other factors, rIFN-beta 2/B cell stimulatory factor 2 (BSF-2)/IL-6 stimulates the growth of human B cells activated by EBV in a manner similar to that induced by activated monocyte supernatants. In addition, an antiserum to IFN-beta that recognizes both IFN-beta 1 and IFN-beta 2 completely neutralizes the B cell growth factor activity of activated monocyte supernatants. These findings demonstrate that IFN-beta 2/BSF-2/IL-6 is a growth factor for human B cells activated by EBV and suggest that this molecule is responsible for B cell growth stimulation induced by activated monocyte supernatants. We have examined the possibility that IFN-beta 2/BSF-2/IL-6 might also be responsible for B cell growth stimulation by supernatants of EBV-immortalized B cells and thus may function as an autocrine growth factor. However, IFN-beta 2/BSF-2/IL-6 is not detectable in supernatants of EBV-immortalized B cells by immunoprecipitation. Also, an antiserum to IFN-beta that neutralizes IFN-beta 2/BSF-2/IL-6 fails to neutralize autocrine growth factor activity. This suggests that autocrine growth factors produced by EBV-immortalized B cells are distinct from IFN-beta 2/BSF-2/IL-6. Thus, the continuous proliferation of EBV-immortalized B cells is enhanced by either autocrine or paracrine growth factors. One of the mediators with paracrine growth factor activity is IFN-beta 2/BSF-2/IL-6.

Monocyte-derived human B-cell growth factor identified as interferon-beta 2 (BSF-2, IL-6).

Soluble products of either Epstein-Barr virus (EBV)-infected B cells or activated monocytes promote the proliferation of EBV-infected B cells and permit their growth at low cell densities. This suggests that growth factors are important for B-cell immortalization by EBV. In this study, a monocyte-derived factor that promotes the growth of EBV-infected b cells was purified and identified as interferon-beta 2 (IFN-beta 2), which is also known as 26-kilodalton protein, B-cell differentiation factor (BSF-2), and interleukin-6 (IL-6). The purified protein has a specific activity of approximately 4 X 10(7) units per milligram of protein in assays of B-cell growth. Thus, IFN-beta 2/BSF-2 is a B-cell growth factor that promotes the proliferation of human B cells infected with EBV.