PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation.

VEGF induces normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. This transition depends on the balance between VEGF-induced endothelial stimulation and PDGF-BB-mediated pericyte recruitment, and co-expression of PDGF-BB normalizes aberrant angiogenesis despite high VEGF doses. We recently found that VEGF over-expression induces angiogenesis in skeletal muscle through an initial circumferential vascular enlargement followed by longitudinal splitting, rather than sprouting. Here we investigated the cellular mechanism by which PDGF-BB co-expression normalizes VEGF-induced aberrant angiogenesis. Monoclonal populations of transduced myoblasts, expressing similarly high levels of VEGF alone or with PDGF-BB, were implanted in mouse skeletal muscles. PDGF-BB co-expression did not promote sprouting and angiogenesis that occurred through vascular enlargement and splitting. However, enlargements were significantly smaller in diameter, due to a significant reduction in endothelial proliferation, and retained pericytes, which were otherwise lost with high VEGF alone. A time-course of histological analyses and repetitive intravital imaging showed that PDGF-BB co-expression anticipated the initiation of vascular enlargement and markedly accelerated the splitting process. Interestingly, quantification during in vivo imaging suggested that a global reduction in shear stress favored the initiation of transluminal pillar formation during VEGF-induced splitting angiogenesis. Quantification of target gene expression showed that VEGF-R2 signaling output was significantly reduced by PDGF-BB co-expression compared to VEGF alone. In conclusion, PDGF-BB co-expression prevents VEGF-induced aberrant angiogenesis by modulating VEGF-R2 signaling and endothelial proliferation, thereby limiting the degree of circumferential enlargement and enabling efficient completion of vascular splitting into normal capillary networks despite high VEGF doses.

Macrophage-mediated angiogenic activation of outgrowth endothelial cells in co-culture with primary osteoblasts.

The successful vascularisation of complex tissue engineered constructs for bone regeneration is still a major challenge in the field of tissue engineering. In this context, co-culture systems of endothelial cells and osteoblasts represent a promising approach to advance the formation of a stable vasculature as well as an excellent in vitro model to identify factors that positively influence bone healing processes, including angiogenesis. Under physiological conditions, the activation phase of angiogenesis is mainly induced by hypoxia or inflammation. Inflammatory cells such as macrophages secrete proinflammatory cytokines and proangiogenic growth factors, finally leading to the formation of new blood vessels. The aim of this study was to investigate if macrophages might positively influence the formation of microvessel-like structures via inflammatory mechanisms in a co-culture system consisting of human outgrowth endothelial cells (OECs) and primary osteoblasts. Treatment of co-cultures with macrophages (induced from THP-1) resulted in a higher number of microvessel-like structures formed by OECs compared to the co-culture. This change correlated with a significantly higher concentration of the proangiogenic VEGF in cell culture supernatants of triple-cultures and was accompanied by an increase in the expression of different proinflammatory cytokines, such as IL-6, IL-8 and TNFα. In addition, the expression of E-selectin and ICAM-1, adhesion molecules which are strongly involved in the interaction between leukocytes and endothelial cells during the process of inflammation was also found to be higher in triple-cultures compared to the double co-cultures, documenting an ongoing proinflammatory stimulus. These results raise the possibility of actively using pro-inflammatory stimuli in a tissue engineering context to accelerate healing mechanisms.

High-dose chemotherapy shows a dose-dependent toxicity to bone marrow osteoprogenitors: a mechanism for post-bone marrow transplantation osteopenia.

BACKGROUND: Osteoporosis is a sequela of hemopoietic cell transplantation with a complex multifactorial pathogenesis in which the relative role of chemotherapy and irradiation is not completely understood. Therefore, the authors investigated the toxicity of chemotherapy-only conditioning regimens on bone homeostasis and bone marrow osteoprogenitors, its dose dependency, and the mechanism of chemotherapy-induced osteopenia. METHODS: Fifty-one patients with high-grade non-Hodgkin lymphoma or breast carcinoma who had been treated previously with high-dose + peripheral blood progenitor cell or conventional chemotherapy or who had not received any treatment (prechemotherapy) were enrolled. The authors measured the bone marrow colony-forming unit fibroblast (CFU-f) and long-term culture-initiating cell frequency, forearm bone mineral density, serum osteotropic hormones and metabolic markers of bone formation (plasma osteocalcin), and resorption (urinary collagen I C-crosslinks). RESULTS: Both high-dose chemotherapy regimens caused a 50% reduction in CFU-f frequency, independently of gonadal function status, whereas conventional chemotherapy and prechemotherapy groups were unaffected. Bone mineral density was measured in 26 non-Hodgkin lymphoma patients and again only high-dose chemotherapy caused a 10% loss in cortical bone and 20% in trabecular bone. No endocrine abnormality was found except for the secondary amenorrhea uniformly induced in the high-dose chemotherapy group. In these patients, plasma osteocalcin unexpectedly failed to increase in response to the menopausal increase in bone resorption rate, showing a selective impairment of the osteoblast compartment to cope with increased functional demand. CONCLUSIONS: Chemotherapy without irradiation shows a dose-dependent toxicity to bone marrow stromal osteoprogenitors and can cause osteopenia by direct damage of the osteoblastic compartment, as a mechanism distinct from and summable to hypogonadism.

Bone marrow stromal damage after chemo/radiotherapy: occurrence, consequences and possibilities of treatment.

High dose chemotherapy (CT) followed by bone marrow transplant (BMT) is increasingly used for the treatment of both hematological and solid neoplasms, but an understanding of its late consequences on the marrow microenvironment is still only at its beginning. It is in fact known that marrow stroma is damaged by high-dose cytotoxic therapy and by radiation exposure. However little is known on the extent of this damage and on the self-repair ability of the stroma. The damage of the stromal microenvironment affects the long-term stem cell engraftment and the maintenance of hemopoietic functions. Furthermore, marrow stroma also represents a progenitor compartment for endosteal osteoblasts, and therefore its damage implies alterations of bone metabolism. Indeed, osteoporosis has recently been recognized as a consequence, of BMT, but only a few studies have been performed to establish the functional status of the stromal compartment after treatment with cytotoxic drugs with or without total body irradiation (TBI) and its role in post-BMT sequelae.

Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy.

Bone marrow stromal cells (BMSC) are an attractive target for novel strategies in the gene/cell therapy of hematologic and skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. We investigated the effects of in vitro expansion on BMSC pluripotentiality, proliferative ability, and bone-forming efficiency in vivo. BMSC from three marrow donors were cultured to determine their growth kinetics. At each passage, their differentiation potential was verified by culture in inductive media and staining with alizarin red, alcian blue, or Sudan black, and by immunostaining for osteocalcin or collagen II. First passage cells were compared to fresh marrow for their bone-forming efficiency in vivo. Stromal cell clones were isolated from five donors and characterized for their multidifferentiation ability. The lifespan and differentiation kinetics of five of these clones were determined. After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Experiments on the clones yielded comparable results. Culture expansion causes BMSC to gradually lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion.

Randomized trial of four vs. seven days of ceftriaxone treatment for bacterial meningitis in children with rapid initial recovery.

BACKGROUND: Seven days or more of antimicrobial treatment is the standard for bacterial meningitis, although third generation cephalosporins are usually able to sterilize cerebrospinal fluid within 24 h. The limited experience from shorter regimens in children is encouraging, and we hypothesized that in rapidly recovering patients older than 3 months of age it would pose no risk for adverse outcome. METHODS: Strict clinical and laboratory criteria were used to define rapid initial recovery, in which case ceftriaxone therapy was either stopped after 4 days (4 injections) in children born on even dates (N = 53) or continued for 7 days in patients born on odd dates (N = 47). Outcomes were compared on Day 7 of hospitalization and at 1 to 3 months after discharge. RESULTS: On Day 7 no differences (P > 0.05 for each criteria) were observed between the 4-day and the 7-day groups regarding fever, clinical signs or serum C-reactive protein concentration. At the follow-up visit 1 to 3 months after discharge the 4-day group had fewer sequelae than the 7-day group (0% vs. 5% neurologic sequelae, P = 0.39 and 3% vs. 9% hearing loss, P = 0.49, respectively). One child in the 4-day group who had fully recovered was subsequently readmitted 53 days after the first hospitalization with recurrent Haemophilus influenzae meningitis. CONCLUSIONS: Four days of ceftriaxone therapy proved to be a safe alternative in patients with rapid initial recovery from bacterial meningitis. A 4-day course of treatment is particularly beneficial for countries with limited resources.

Chondrocyte and osteoblast differentiation stage-specific monoclonal antibodies as a tool to investigate the initial bone formation in developing chick embryo.

Taking advantage of monoclonal antibodies raised against chick hypertrophic chondrocytes (BD5, LA5, AD2) and osteoblasts (SB1, SB2, SB3, SB5), we have investigated expression of differentiation stage-specific antigens by further differentiating hypertrophic chondrocytes. Expression of all antigens was developmentally regulated during in vitro further differentiation of hypertrophic chondrocytes and all monoclonal antibodies, including those raised against osteoblasts, recognized antigens synthesized by chondrocytes at specific differentiation stages. In particular, the expression of the SB5 antigen, considered specific for secretory osteoblasts incorporated into the bone matrix, progressively increased during the chondrocyte culture and reached its maximum at the time of matrix mineralization. Interestingly, the SB3 antigen was transiently expressed at an intermediate phase of the culture with a staining pattern characteristic of cell-cell adhesion proteins. When cryosections of the chondro-bone junction of developing embryo tibiae were stained, it was observed that monoclonal antibodies raised against hypertrophic chondrocytes stained frank hypertrophic and elongated chondrocytes (BD5, LA5), a very discrete peripheric layer of cartilage (AD2), and cells embedded in the initial osteoid matrix (LA5), but failed to stain cells in the preosteoblast and osteoblast region. On the contrary, monoclonal antibodies recognizing antigens on the surface of cells in the osteogenic layer surrounding the initial bone, also stained frank hypertrophic and/or elongated chondrocytes on the cartilage side of the initial bone (SB1, SB2, SB3). The SB5 monoclonal antibody, in addition to staining cells in the osteoid, also stained a few chondrocytes immediately adjacent. These observations support the concept that hypertrophic chondrocytes may further differentiate to osteoblast-like cells and participate to the initial bone formation.

C-reactive protein in measles.

Seventy-two children with early measles (1st-3rd day of rash), presenting at two centres in Santiago, Chile, were classified as having mild ('ordinary measles', n = 50), or moderate to severe measles ('primarily severe measles', n = 22). The level of serum C-reactive protein (CRP) was determined by nephelometry from a finger prick sample. The mean CRP value in ordinary measles, 19 mg/l, was significantly lower (P less than 0.001) than in primarily severe measles where the mean CRP was 65 mg/l. During late measles (5th-8th day of rash), the mean CRP was 19 mg/l if the child recovered uneventfully (n = 35), whereas the mean level of 123 mg/l (P less than 0.001) was encountered when the child suffered from complicating pneumonia (n = 22). We conclude that the simple quantitative CRP determination is a useful alarm signal during the course of measles: elevated levels point to severity or complications in recovery.

Early stage Hodgkin's disease: ten-year results of a non-randomised study with radiotherapy alone or combined with MOPP.

From September 1976 to June 1982, 201 consecutive patients with stage I (A and B)-IIA Hodgkin's disease were stratified in two groups according to prognostic factors. The F group included 116 patients with favourable presentation: they were staged with laparotomy and treated with subtotal or total nodal radiotherapy alone. The U group included 85 cases with unfavourable presentation who were staged by laparoscopy and treated with 3MOPP (mechlorethamine, vincristine, procarbazine, prednisone)-radiotherapy-3MOPP. At 10 years the F group showed a freedom from progression (FFP) of 71% with significant difference between stage I and II (85% vs. 59%; P = 0.003) and an overall survival of 84%. The results of the U group were: FFP 83%, overall survival 74%, and the findings were not influenced by stage. FFP in patients with bulky vs. not bulky lymphoma was 70% vs. 87% (P = 0.04). No secondary acute non-lymphocytic leukaemia developed among patients treated with radiotherapy and in continuous complete remission, while acute leukaemia occurred in the F group patients who received salvage chemotherapy (4 of 31 cases) and in the U group (3 of 85 cases). Present results confirm the usefulness of radiotherapy alone in favourable pathological stage IA. All other disease stages will require a different strategy that should consist of radiotherapy combined with short-term effective regimens, such as ABVD (doxorubicin, bleomycin, vinblastine and decarbazine) or VBM (vinblastine, bleomycin and methotrexate) to reduce the incidence of MOPP-associated gonadal dysfunction and leukaemogenesis.

Serum C-reactive protein in childhood meningitis in countries with limited laboratory resources: a Chilean experience.

Quantitative C-reactive protein (CRP) was determined sequentially by nephelometry and photometry from a finger prick serum sample in 67 children with bacterial meningitis (BM) and 16 children with aseptic meningitis (AM). The initial mean CRP value of 180 mg/liter in children with BM differed significantly from the 12 mg/liter found in those with AM (P less than 0.001). In BM a slow descent instead of rapid normalization or a secondary increase in sequential CRP values were early indicators of complications during recovery, such as resistance to the antibiotic. A significant difference in the mean CRP values between uneventful and complicated courses of BM was observed from the fourth day on (P less than 0.001). The measurements obtained with nephelometry correlated reliably with the more widely available photometry (r = 0.99). Easily performed rapid CRP determinations can considerably improve the quality of care in meningitis patients, especially in those situations where facilities for performing bacterial cultures or antibiotic susceptibility testing are not available.

Malonamic acid derivatives as M1 selective muscarinic receptor antagonists.

A series of malonamic acid esters with suitable amino alcohols, typical of antimuscarinic compounds, was synthesized and the affinities for the three pharmacologically defined muscarinic receptor subtypes, namely M1, M2 and M3, were evaluated by radioligand displacement experiments. It was found that the esters with 3-quinuclidinol 7b, 7f-g, 8 and 9 are ligands with intermediate to high affinity for the M1 receptors, for which they show a preferential binding. Unexpectedly, the ester 7a with tropine bound with negligible affinity to all the receptors investigated. The introduction of a phenyl group on the carboxamido moiety of 7b gave compound 9, which showed an affinity for the M1 receptor comparable with that of the reference drug Pirenzepine 1.

Conservative treatment of early breast cancer. Long-term results of 1232 cases treated with quadrantectomy, axillary dissection, and radiotherapy.

One thousand two hundred and thirty-two women with invasive breast cancer lesions measuring less than 2 cm in diameter, clinically assessed as T1N0-1M0, were treated from 1970 to 1983 at the National Cancer Institute of Milan with quadrantectomy, axillary dissection, and radiotherapy (QUART). Pathologic evidence of lymph-nodes metastases was found in 32% of the patients. Overall survival at 5 and 10 years from surgery was 91% and 78%, respectively. The cumulative probability of survival tends to decrease with increasing tumor size: the 7-year survival rate was 84% in cases in which lesions measured from 1.6 to 2.0 cm, and 94% in cases in which the lesions were less than 0.5 cm. Tumor site in the treated breast did not affect distant outcome. No difference was found between the patients without node metastases and patients with one node involved, whereas the patients with more than one node showed a lower probability of survival. The survival curves of 352 cases treated inside a randomized trial and that of 880 cases routinely treated appear to be superimposable. Local recurrences and new primary ipsilateral tumors were, respectively, 35 (2.8%) and 19 (1.6%); 56 women with local recurrences or second tumors underwent second surgery (total mastectomy, 43; wide resection, 11). Five of them died from distant spread of breast cancer, while 49 are alive and well. In the contralateral breasts 45 carcinomas were recorded during the follow-up time. The results of the present analysis of a large number of T1 cases reconfirm the safety of integrated radiosurgical conservative treatments.

Breast conservation is the treatment of choice in small breast cancer: long-term results of a randomized trial.

From 1973 to 1980, 701 women with small breast cancer (less than 2 cm in diameter) were randomized into two different treatments. 349 patients received classic Halsted mastectomy and 352 patients received quadrantectomy, axillary dissection and radiotherapy on the ipsilateral breast. 24.6% of the patients in the mastectomy group and 27.0% of the patients in the conservation group had axillary metastases. Overall 10 year survival was 76% in the Halsted patients and 79% in the quadrantectomy patients; 13 year survival was 69% and 71%, respectively. No differences were observed after analysis by site and size of the primary tumour and age of the patients. Patients with positive axillary nodes had consistently better survival curves in the quadrantectomy group compared with the Halsted group (not significant). Among the quadrantectomy patients there were 11 local recurrences (with 4 deaths) while among the Halsted patients, 7 had local recurrences (5 deaths). There were 19 cases of contralateral breast carcinomas in the quadrantectomy group and 20 in the Halsted group. At 16 years from the beginning of the trial no evidence of oncogenic radiation risk was observed. In patients with small size carcinomas total mastectomy should have no role.

Hodgkin's disease: the Milan Cancer Institute experience with MOPP and ABVD.

The report includes the essential therapeutic and toxic results following MOPP vs. MOPP/ABVD and MOPP vs. ABVD within a combined modality setting. The findings indicate that in stage IV the alternating regimen is superior to MOPP while in stage IIB-III ABVD plus radiotherapy yielded superior results associated with no treatment-induced sterility and leukemia compared with MOPP plus radiotherapy.

Combined modality treatment for stage I-II non-Hodgkin's lymphomas: CVP versus BACOP chemotherapy.

This paper reports the 5-year results of a prospective randomized study beginning in 1976 on 177 evaluable patients with pathologic Stage I-IE and II-IIE non-Hodgkin's lymphomas with diffuse histology according to the Rappaport classification. Treatment consisted of either CVP or BACOP chemotherapy (3 cycles) followed by regional radiotherapy (40 to 50 Gy) and further cycles of either combination. In both arms, complete remission at the end of combined treatment was high (CVP 93%, BACOP 98%) regardless of age, stage or bulky disease. At 5 years, the comparative freedom from first progression was 62% for CVP vs 78% for BACOP (p = 0.02), respectively. Clinically relevant differences favoring BACOP chemotherapy were essentially documented in patients with large cell lymphomas (International Working Formulation), those with Stage II having more than three involved anatomical sites, bulky disease and age over 60 years. Recurrence within radiation fields was documented in only 5% of complete responders. Combined treatment was, in general, well tolerated particularly when BACOP was used. In only 2 patients given CVP post radiation cutaneous fibrosis was documented. Second solid tumors were detected in 4 patients. One patient started on CVP died because of brain stem necrosis after 45 Gy. We conclude that in Stage I-II patients with nodal and extranodal diffuse non-Hodgkin's lymphomas, particularly large cell lymphomas, combined modality approach with primary Adriamycin and bleomycin containing regimen, such as BACOP, followed by adjuvant radiotherapy offers high chances of cure with minimal toxicity.

Contralateral breast cancer after limited surgery plus radiotherapy of early mammary tumors.

Conservative treatment of early breast cancer with limited surgery requires a mandatory irradiation of the affected breast, which implies a low but measurable irradiation of contralateral breast too. As ionizing radiations can produce an oncogenic effect on mammary tissues, the series of 701 patients of the Milano clinical trial on T1 No breast cancer (1973-1980) was investigated to compare the incidence of contralateral breast cancer in the Halsted group (not irradiated) and in the QU.A.RT. group (irradiated on the operated breast with a total dose of 50 Gy plus a limited boost of 10 Gy). In March 1986, a contralateral breast cancer was diagnosed in 17/349 patients (4.9%) of the Halsted group and in 18/352 patients (5.1%) of the QU.A.RT. group after a median follow-up of 108 months. The sites of contralateral cancer were superimposable in the two groups of patients, with a constant prevalence of external quadrants, despite the great difference of dose distribution in the irradiated patients. Our data on the incidence of contralateral breast cancer failed to demonstrate an oncogenic effect of irradiation to date, but the follow-up is still in progress and any future event will be registered and discussed.

Germ cell tumors of the ovary: the experience of the National Cancer Institute of Milan. I. Dysgerminoma.

The experience of the Istituto Nazionale Tumori of Milan on dysgerminoma is presented. Between 1970 and December of 1982, 25 patients were treated with a unique protocol which considered surgery and radiotherapy with different schedules according to the extension of the disease. With this treatment protocol all 13 patients at Stage I were alive and free of disease with a median follow-up of 77 months. Of 12 patients at Stage III (10 retroperitoneal and 2 retroperitoneal and peritoneal) 4 relapsed. The 5-year relapse-free survival of Stage III patients was 61.4% and the overall survival 89.5%. Amenorrhea due to radiation dose absorbed by the contralateral shielded ovary was found in 7.7%. The excellent results in Stage I patients were balanced by the unsatisfactory results in Stage III patients. A more aggressive treatment and the knowledge of other prognostic factors seem necessary.

Comparison of Halsted mastectomy with quadrantectomy, axillary dissection, and radiotherapy in early breast cancer: long-term results.

From 1973 to 1980 we randomly assigned 701 patients with breast cancer measuring less than 2 cm in diameter and with no palpable axillary lymph nodes to Halsted radical mastectomy (n 349) or to 'quadrantectomy' with axillary dissection and radiotherapy to the ipsilateral breast tissue (n 352). The two groups were comparable in age distribution, size and site of primary tumor, menopausal status, and frequency of axillary metastases. The average follow-up time was 103 months. Actuarial curves show no difference between the two groups in the disease-free interval after surgery or in the overall survival rate. At 8 yr the disease-free survival was 77% for the patients in the Halsted group and 80% for those in the 'quadrantectomy' group, and the overall survival was 83 and 85% respectively. We conclude that small breast cancers may be safely treated with the conservative treatment described. In our opinion total ablative operations are not justified.