Operation of the Preclinical Head Scanner for Proton CT.

We report on the operation and performance tests of a preclinical head scanner developed for proton computed tomography (pCT). After extensive preclinical testing, pCT is intended to be employed in support of proton therapy treatment planning and pre-treatment verification in patients undergoing particle-beam therapy. In order to assess the performance of the scanner, we have performed CT scans with 200 MeV protons from both the synchrotron of the Loma Linda University Medical Center (LLUMC) and the cyclotron of the Northwestern Medicine Chicago Proton Center (NMCPC). The very high sustained rate of data acquisition, exceeding one million protons per second, allowed a full 360° scan to be completed in less than 7 minutes. The reconstruction of various phantoms verified accurate reconstruction of the proton relative stopping power (RSP) and the spatial resolution in a variety of materials. The dose for an image with better than 1% uncertainty in the RSP is found to be close to 1 mGy.

200 MeV proton radiography studies with a hand phantom using a prototype proton CT scanner.

Proton radiography has applications in patient alignment and verification procedures for proton beam radiation therapy. In this paper, we report an experiment which used 200 MeV protons to generate proton energy-loss and scattering radiographs of a hand phantom. The experiment used the first-generation proton computed tomography (CT) scanner prototype, which was installed on the research beam line of the clinical proton synchrotron at Loma Linda University Medical Center. It was found that while both radiographs displayed anatomical details of the hand phantom, the energy-loss radiograph had a noticeably higher resolution. Nonetheless, scattering radiography may yield more contrast between soft and bone tissue than energy-loss radiography, however, this requires further study. This study contributes to the optimization of the performance of the next-generation of clinical proton CT scanners. Furthermore, it demonstrates the potential of proton imaging (proton radiography and CT), which is now within reach of becoming available as a new, potentially low-dose medical imaging modality.

The growth curve of the first passage xenografts of human tumors in nude mice predicts their transplantation behaviour.

The patterns of macroscopic growth of 20 first generation human tumor xenografts (16 renal cell carcinomas and 4 squamous cell carcinomas of the head and neck region) were studied with the recursion formula of the Gompertz function. This method enables the characterization of each tumor growth pattern by two parameters: (i) parameter a, which correlates with the starting growth rate of the tumor and (ii) parameter b, which is a measure of the intensity of growth deceleration as a function of tumor growth. A statistically significant prediction of the establishment of a xeno-transplantation line after serial subpassaging (in 9/20 tumors) according to the characteristics of first passage growth curves (p<0. 01) is reported. Especially parameter b, the value of the growth deceleration, highly correlates with serial growth: tumors showing less growth deceleration (higher b-values) during first passage more frequently develop into transplantation lines. On the contrary line development could not be predicted on the basis of the a-values of the first passage growth curves alone (p=0.137). This observation adds to the accumulating evidence, that the process of tumor growth deceleration is a pivotal parameter of tumor biology. Moreover, the present evaluation substantially reduces the time needed to assay serial growth of tumor xenografts for prognostic purposes making this assay potentially more attractive for clinical use.

Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.

Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV.

Prognostic relevance of the intrinsic growth deceleration of the first passage xenografts of human renal cell carcinomas.

BACKGROUND: Although successful xenotransplantation of human tumors in nude mice highly predicts prognosis, little is known regarding the biologic background of this correlation. In this study, the relationship between the macroscopic growth pattern of first-generation xenografts of human renal cell carcinomas in nude mice and prognosis was studied. METHODS: Macroscopic growth patterns of the first-generation xenografts of locally confined renal cell carcinomas were analyzed according to the best-fit Gompertz recursion formulas. RESULTS: The parameter "b" of the growth pattern, the measure of the intensity of growth deceleration as a function of tumor growth, strongly predicted prognosis after nephrectomy as a single factor; faster growth deceleration, i.e., lower b values, predicted better prognosis (mean follow-up, 5.2 years; P = 0.000008 for the disease free period and P = 0.000018 for overall survival). It is also the most significant single prognostic parameter among others (including staging and grading) according to a multivariate analysis. CONCLUSIONS: The parameter expressing the Gompertzian macroscopic growth deceleration of the first-generation xenografts of clinically locally confined renal cell carcinomas in nude mice explains the strong prognostic impact of xenotransplantation.

Virus entry into a polarized epithelial cell line (MDCK): similarities and dissimilarities between influenza C virus and bovine coronavirus.

We have analysed the uptake of influenza C virus and bovine coronavirus (BCV) by a polarized epithelial cell line, Madin-Darby canine kidney (MDCK) cells. Both viruses use N-acetyl-9-O-acetylneuraminic acid as a receptor determinant for attachment to cells. Virus binding assays with immobilized proteins indicated that a glycoprotein of 40 kDa is the major surface protein containing the receptor determinant for the two viruses. MDCK cells grown on filters for permeable support were found to have differential sensitivity to infection by these viruses. Both viruses were able to initiate infection via the apical domain of the plasma membrane, but only influenza C virus also accomplished infection via the basolateral plasma membrane. The resistance of MDCK cells to BCV infection from the basal filter chamber was overcome when the cell polarity was abolished by maintaining the cells in calcium-free medium. This finding indicates that the resistance to basolateral infection by BCV is a property of the cell line and not due to a technical problem related to the use of filters. Our results indicate that two viruses which use the same receptor for attachment to cells may differ in their ability to enter polarized cells. The possible involvement of an accessory molecule in the entry of BCV is discussed.

Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuraminic acid) binding activity.

The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of alpha-2,3-linked sialic acid on the erythrocyte surface. N-Glycolylneuraminic acid was recognized more efficiently by TGEV than was N-acetylneuraminic acid. For an efficient hemagglutination reaction the virions had to be treated with sialidase. This result suggests that the sialic acid binding site is blocked by virus-associated competitive inhibitors. Porcine respiratory coronavirus (PRCV), which is serologically related to TGEV but not enteropathogenic, was found to be unable to agglutinate erythrocytes. Incubation with sialidase did not induce a hemagglutinating activity of PRCV, indicating that the lack of this activity is an intrinsic property of the virus and not due to the presence of competitive inhibitors. Only monoclonal antibodies to an antigenic site that is absent from the S protein of PRCV were able to prevent TGEV from agglutinating erythrocytes. The epitope recognized by these antibodies is located within a stretch of 224 amino acids that is missing in the S protein of PRCV. Our results indicate that the sialic acid binding activity is also located in that portion of the S protein. The presence of a hemagglutinating activity in TGEV and its absence in PRCV open the possibility that the sialic acid binding activity contributes to the enterotropism of TGEV.

Significance of conventional and new prognostic factors for locally confined renal cell carcinoma.

BACKGROUND: The prognosis of patients with locally confined renal cell carcinoma is variable. To improve the prognostic knowledge and select patients at high risk, additional prognostic parameters are needed. METHODS: The significance with respect to survival and tumor recurrence of "classic" and "new" prognostic parameters has been examined by following 41 patients with locally confined renal cell carcinoma after nephrectomy (mean follow-up, 5.2 years). The significance of histologic grade, tumor stage, Ki-67 index, proliferating cell nuclear antigen index, 3H-thymidine labeling index, tumor ploidy status, and tumor growth after xenotransplantation into nude mice (GAX range) was tested using the Kaplan-Meier plots by the log rank test or Tarone's test and also by the Cox multiple hazard regression analysis. RESULTS: Tumor stage (P < 0.0025), histologic grade (P < 0.005), Ki-67 index (P < 0.006), and GAX range (P < 0.00004) were found to be significant prognostic parameters for survival and tumor recurrence using single-factor analysis. Applying the multivariate analysis, the combination of the "new" factors, GAX range and Ki-67 index, resulted in even a higher prognostic relevance than the combination of the "classic" prognostic factors, tumor stage and histologic grade. The calculated prognostic index based on the results of the Cox analysis, which, except for stage and grade, included the Ki-67 index, was shown to be highly correlated with survival (P = 0.00002) and tumor recurrence (P = 0.0004). Its prognostic validity was studied with the receiver operating characteristics procedure and was found to be considerably superior to that of the two conventional prognosticators. CONCLUSIONS: The additional determination of the Ki-67 labeling index increases the prognostic assessment of patients with locally confined renal cell carcinoma.

Effect of biological response modifiers on growth and cell proliferation of human tumor xenografts in nude mice.

The effect of biological response modifiers on macroscopic tumor growth and on tumor cell proliferation of a human renal cell carcinoma and a squamous cell carcinoma (hypopharynx) in nude mice has been studied. Tumor necrosis factor alpha (TNF-alpha) and interferon alpha (IFN-alpha) as well as granulocyte-macrophage colony-stimulating factor (GM-CSF) were applied either alone or in combination, and TNF-alpha was also combined with etoposide (ETP). TNF-alpha and IFN-alpha alone or in combination did not substantially affect the course of tumor growth, however, they did influence the pattern of tumor growth. There was also only a marginal effect on tumor cell proliferation. However, IFN-alpha protects the animals from tumor growth associated weight loss. ETP and ETP plus TNF-alpha leads to a deceleration of tumor growth, a decrease of the labeling index and to a significant decrease of the animal weight which indicates that the first two effects may be partly due to the toxicity of the treatment. GM-CSF modifies cell proliferation in a dose-dependent manner, i.e. stimulation at low doses and tendency to inhibition at higher doses. Although there is no substantial direct antineoplastic effect of the agents studied, the results make clear that indirect effects of therapeutic agents due to therapy induced cachexia should always be regarded. It is interesting that IFN-alpha has a protective effect against cachexia.

Analysis of the sialic acid-binding activity of the transmissible gastroenteritis virus.

Porcine transmissible gastroenteritis virus (TGEV) has been shown to agglutinate erythrocytes using alpha 2,3-linked sialic acid on the cell surface as binding site. The hemagglutinating activity requires the pretreatment of virus with neuraminidase. We obtained evidence that TGEV recognizes not only N-acetylneuraminic acid but also N-glycoloylneuraminic acid, a sialic acid present on many porcine cells.

Analysis of cellular receptors for human coronavirus OC43.

Bovine coronavirus (BCV), human coronavirus OC43 (HCV-OC43) and hemagglutinating encephalomyelitis virus (HEV) are serologically related viruses that all have hemagglutinating activity. The receptor determinant for attachment to erythrocytes has been shown to be N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). We compared the ability of the three coronaviruses to recognize 9-O-acetylated sialic acid and found that they all bind to Neu5,9Ac2 attached to galactose in either A2,3 or A2,6-linkage. There are, however, some differences in the minimum amount of sialic acid that is required on the cell surface for agglutination by these viruses. Evidence is presented that HCV-OC43 uses Neu5,9Ac2 as a receptor determinant not only for agglutination of erythrocytes but also for attachment to and infection of a cultured cell line, MDCK I cells.

Polarized entry of bovine coronavirus in epithelial cells.

Epithelial cells are highly polarized cells divided into an apical and a basolateral plasma membrane. The two domains are composed of a distinct set of proteins and lipids. Concerning virus infection of epithelial cells, the polarity of host cell receptor distribution defines the domain from which infection may be mediated. We were interested to analyze the infection of polarized cells by bovine coronavirus (BCV). The entry of BCV into MDCK I cells was investigated by growing the cells on a permeable support. Cell were infected with BCV from either the apical or basolateral domain. The efficiency of infection was determined by measuring the hemaglutinating activity of the virus released into the apical compartment. Virus replication was only detectable after inoculation from the apical surface. Therefore, infection of MDCK I cells with BCV is restricted to the apical side.

Recognition of cellular receptors by bovine coronavirus.

Bovine coronavirus (BCV) initiates infection by attachment to cell surface receptors the crucial component of which is N-acetyl-9-O-acetylneuraminic acid. Inactivation of receptors by neuraminidase treatment and restoration of receptors by enzymatic resialylation of asialo-cells is described as a method to determine (i) the type of sialic acid that is recognized; (ii) the linkage specificity of the viral binding activity; (iii) the minimal amount of sialic acid required for virus attachment. Evidence is presented that both glycoproteins and glycolipids can serve as receptors for BCV provided they contain 9-O-acetylated sialic acid. A model is introduced proposing that after initial binding to sialic acid-containing receptors, the S-protein of BCV interacts with a specific protein receptor. This interaction may result in a conformational change that exposes a fusogenic domain and thus induces the fusion between the viral and the cellular membrane.

Treatment with tumor necrosis factor alpha and interferon alpha of a human kidney cancer xenograft in nude mice: evidence for an anticachectic effect of interferon alpha.

Unfortunately the efficacy of the treatment of the metastatic or recurrent renal cell carcinoma (RCC) has not improved during the last few years. Recently effort has been put into the experimental and clinical evaluation of so-called "biological response modifiers" (BRM; cytokines and related peptides) as treatment modalities for RCC. The present results are, however, still disappointing. Since BRM, if applied alone, are largely ineffective as antineoplastic agents, more experimental studies are now necessary to test the antineoplastic value of their combinations, which seem to be more promising. In the present study, the in vivo effect of tumor necrosis factor a (TNF a) and/or interferon a (IFN a) on the macroscopic tumor growth (external caliper measurements of tumor size) and on the cell proliferation (in vivo 3H-thymidine labelling index, LI, and mitotic index, MI) of a human RCC xenograft line in nude mice has been investigated. Neither of these substances alone nor their combination was effective in changing the time course of the tumor sizes and the growth patterns of the treated tumors in a statistically significant manner as compared to the untreated controls. Also the cell kinetic parameters were only marginally affected by these treatments, whereby TNF a alone proved to be more effective than IFN a alone. However, compared to the effect of TNF alpha alone, the combination with IFN alpha leads to some amelioration of the cell kinetic perturbations and also to an appreciable shift in the growth patterns of the tumors from distinct Gompertzian (under TNF alpha alone) to near exponential (under the combination treatment; p < 0.05). As a consequence, the tumors grow more slowly under the combined treatment during the observation time, and on the other hand, their growth does not decelerate as much as under TNF alpha alone. Actually, if tumor growth continues in the same way, the extrapolation of the present data predicts smaller and greater tumors than the control tumors in the TNF alpha and in the combination treatment groups respectively. Notably, in the combination the effect of the IFN alpha seems to predominate. This is also seen in the effect of this combination on the cachexia of these tumor-bearing animals: either alone or in combination with TNF alpha, IFN alpha partially protects the animals from tumor-growth associated weight loss. Although the direct antineoplastic in vivo effect of the present cytokine combination against this human RCC xenograft line is rather limited, the potential antagonizing effect of IFN alpha on the development of cachexia should be further explored.

Treatment of a human renal cell carcinoma in nude mice with recombinant human tumor necrosis factor alpha and etoposide.

The effect of treating a human renal cell adenocarcinoma xenografted into Balb/c-nu/nu (nude) mice with recombinant human tumor necrosis factor alpha (TNF alpha) and the cytostatic agent etoposide (ETP) as monotherapy or combination has been studied. Antitumor effects were evaluated by determining growth of the tumor implants by external caliper measurements and tumor cell proliferation by determining the labelling index (LI) after pulse labelling with 3H-thymidine. The toxicity of the treatment with TNF alpha and/or ETP was also studied by measuring the animal weight. Monotherapy with TNF alpha had no effect on tumor growth or proliferation. Treatment with ETP as a single agent, TNF alpha plus ETP applied concurrently and TNF alpha plus ETP two days later led to a slight inhibition of tumor growth and also to a slight decrease of the LI. In contrast to a monotherapy with TNF alpha, all therapeutic modalities containing ETP showed an increased toxic effect on the animals represented by a distinct weight loss. This suggests that the minute efficacy of the treatment observed could well be due solely to its toxicity. In contrast to two other studies, no additive or synergistic effect of the antineoplastic activity of TNF alpha and/or ETP was found. The intertumoral variation of human renal cell carcinomas could be one reason for the different results with this therapeutic regimen.

Lethal toxicity of cyclophosphamide depends on the tumor stage. Studies on the syngeneic adenocarcinoma EO 771 in C57bl/6j mice.

The effect of tumor size and tumor age on the efficiency, and particularly on the lethal toxicity, of a single application of cyclophosphamide (300 micrograms/g body weight i.p.) to C57bl/6j mice bearing the adenocarcinoma EO 771 has been investigated by treating tumors of the same size but different age on the one hand and tumors of the same age but different size on the other. Treatment of animals bearing larger tumors is consistently associated with increased toxicity: 71/74 animals with tumors > 1.25 g at the time of treatment die of toxicity as opposed to only 1/33 animals with tumors < 1.25 g (relative risk of death of toxicity is 31.7 for animals with tumors > 1.25 g as compared to those bearing tumors < 1.25 g). On the contrary, the age of the tumor does not appreciably influence the toxicity of treatment. The reason for the increased toxicity of the antineoplastic chemotherapy with increasing tumor size is not clear. However, the rather abrupt manifestation of lethal toxicity as a function of tumor size coincides well with the beginning of tumor cachexia in the same animals. Possible mechanisms for this interrelationship of cachexia and potentiation of treatment toxicity are discussed.

Recognition of N-acetyl-9-O-acetylneuraminic acid by bovine coronavirus and hemagglutinating encephalomyelitis virus.

The S protein of hemagglutinating encephalomyelitis virus is shown to be a hemagglutinin requiring N-acetyl-9-O-acetylneuraminic acid as a receptor determinant on the surface of erythrocytes. The ability of bovine coronavirus to recognize 9-O-acetylated sialic acid was used to establish a binding assay for the detection of glycoproteins containing this type of sugar. The assay is very fast, because it uses the acetylesterase of the viral HE protein to localize bound virus.

N-acetylneuraminic acid plays a critical role for the haemagglutinating activity of avian infectious bronchitis virus and porcine transmissible gastroenteritis virus.

Porcine transmissible gastroenteritis virus (TGEV) was found to resemble avian infectious bronchitis virus (IBV) in its interaction with erythrocytes. Inactivation of the receptors on erythrocytes by neuraminidase treatment and restoration of receptors by reattaching N-acetylneuraminic acid (Neu5Ac) to cell surface components indicated that alpha 2,3-linked Neu5Ac serves as a receptor determinant for TGEV as has been reported recently for IBV. Similar to IBV, the haemagglutinating activity of TGEV is evident only after pretreatment of virus with neuraminidase indicating that inhibitors on the virion surface have to be inactivated in order to induce the HA-activity of these viruses. A model is presented to explain why the HA-activity of untreated virus is masked and how neuraminidase treatment results in the unmasking of this activity.

Neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes.

The interaction of infectious bronchitis virus (IBV) with erythrocytes was analyzed. The binding activity of IBV was not sufficient to agglutinate chicken erythrocytes. However, it acquired hemagglutinating activity after treatment with neuraminidase to remove alpha 2,3-linked N-acetylneuraminic acid from the surface of the virion. Pretreatment of erythrocytes with neuraminidase rendered the cells resistant to agglutination by IBV. Susceptibility to agglutination was restored by resialylation of asialo-erythrocytes to contain alpha 2,3-linked sialic acid. These results indicate that IBV attaches to receptors on erythrocytes, the crucial determinant of which is sialic acid alpha 2,3-linked to galactose. In contrast to other enveloped viruses with such a binding specificity (influenza viruses and paramyxoviruses) IBV lacks a receptor-destroying enzyme.

Bovine coronavirus uses N-acetyl-9-O-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells.

The importance of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant for bovine coronavirus (BCV) on cultured cells was analysed. Pretreatment of MDCK I (Madin Darby canine kidney) cells with neuraminidase or acetylesterase rendered the cells resistant to infection by BCV. The receptors on a human (CaCo-2) and a porcine (LLC-PK1) epithelial cell line were also found to be sensitive to neuraminidase treatment. The susceptibility to infection by BCV was restored after resialylation of asialo-MDCK I cells with Neu5,9Ac2. Transfer of sialic acid lacking a 9-O-acetyl group was ineffective in this respect. These results demonstrate that 9-O-acetylated sialic acid is used as a receptor determinant by BCV to infect cultured cells. The possibility is discussed that the initiation of a BCV infection involves the recognition of different types of receptors, a first receptor for primary attachment and a second receptor to mediate the fusion between the viral envelope and the cellular membrane.