Giancarlo Andrighetto

Forecasting the growth of multicell tumour spheroids: implications for the dynamic growth of solid tumours.

The growth dynamics of multicell tumour spheroids (MTS) were analysed by means of mathematical techniques derived from signal processing theory. Volume vs. time trajectories of individual spheroids were fitted with the Gompertz growth equation and the residuals (i.e. experimental volume determinations minus calculated values by fitting) were analysed by fast fourier transform and power spectrum. Residuals were not randomly distributed around calculated growth trajectories demonstrating that the Gompertz model partially approximates the growth kinetics of three‐dimensional tumour cell aggregates. Power spectra decreased with increasing frequency following a 1/fδ power‐law. Our findings suggest the existence of a source of ‘internal’ variability driving the time‐evolution of MTS growth. Based on these observations, a new stochastic Gompertzian‐like mathematical model was developed which allowed us to forecast the growth of MTS. In this model, white noise is additively superimposed to the trend described by the Gompertz growth equation and integrated to mimic the observed intrinsic variability of MTS growth. A correlation was found between the intensity of the added noise and the particular upper limit of volume size reached by each spheroid within two MTS populations obtained with two different cell lines. The dynamic forces generating the growth variability of three‐dimensional tumour cell aggregates also determine the fate of spheroid growth with a strong predictive significance. These findings suggest a new approach to measure tumour growth potential.

Oscillating growth patterns of multicellular tumour spheroids

Abstract. The growth kinetics of 9L (rat glioblastoma cell line) and U118 (human glioblastoma cell line) multicellular tumour spheroids (MTS) have been investigated by non‐linear least square fitting of individual growth curves with the Gompertz growth equation and power spectrum analysis of residuals. Residuals were not randomly distributed around calculated growth trajectories. At least one main frequency was found for all analysed MTS growth curves, demonstrating the existence of time‐dependent periodic fluctuations of MTS volume dimensions. Similar periodic oscillations of MTS volume dimensions were also observed for MTS generated using cloned 9L cells. However, we found significant differences in the growth kinetics of MTS obtained with polyclonal cells. Our findings demonstrate that the growth patterns of three‐dimensional tumour cell cultures are more complex than has been previously predicted using traditional continuous growth models.

Analysis of CIITA encoding AIR-1 gene promoters in insulin-dependent diabetes mellitus and rheumatoid arthritis patients from the Northeast of Italy: Absence of sequence variability

Qualitative and/or quantitative alterations in the expression of the MHC class II molecules affect the onset and maintenance of the immune response and may be the basis of a wide variety of disease states, such as autoimmunity and immunodeficiency.CIITA is a major physiological regulator of the expression of MHC class II genes. The availability of CIITA ap- pears generally essential for MHC class II gene expression, and hence its own transcriptional regulatory mechanisms result of fundamental importance for a correct homeostasis of the immune response. Therefore, it is possible to hypothesize that variability at the CIITA-encoding locus, AIR-1, could constitute an additional source of susceptible traits to autoimmune diseases. Mutations at AIR-1/CIITA promoters could modulate expression of CIITA. Variations in CIITA expression could influence the qualitative and quantitative expression of MHC class II molecules at cell surface. We have analyzed sequence variation at AIR-1/CIITA promoters by PCR-SSCP in 23 IDDM and 30 RA patients compared to a sample of 19 unaffected normal controls and 16 unaffected IDDM family members, for a total of 88 Caucasian subjects from the Northeast of Italy. No sequence difference was found at the four AIR-1/CIITA promoters between autoimmune patients and normal controls. Moreover, the promoters resulted invariant within the entire group of 88 subjects analyzed, comprising patients and controls. This finding suggests a possible selective advantage in maintaining CIITA upstream regulatory sequences invariant.

Comparison between the in vitro activities of in vivo-induced hapten-specific suppressor cells and supernatants of a hapten-specific suppressor hybridoma

A trinitrophenyl (TNP)-specific suppressor hybridoma was obtained by fusing hapten-binding spleen cells (SC) of BALB/c mice 1 week after intravenous (iv) injection of TNP-modified syngeneic lymphocytes with the AKR lymphoma BW5147. The suppressive activity of supernatants from one clone (TNP-44) was compared with that of in vivo-induced TNP-specific suppressor cells. Both the TNP-specific suppressor cells (TsTNP) and the TNP-44 were hapten binding and hapten specific. They suppressed the functional activity of TNP-haptenized T as well as B cells. TNP-44 supernatant also inhibited the proliferation of TNP-modified cells. Using native target cells, both TNP-44 supernatant and the in vivo-induced suppressor cells suppressed the anti-TNP B-cell response to TNP-bound T-dependent soluble or cellular antigens, but not to TNP-lipopolysaccharide (LPS). Furthermore, the function of TNP-specific helper T cells (THTNP) was impaired in the presence of TSTNP or supernatant from TNP-44. From these observations it was concluded that both the TSTNP and a TNP-specific factor derived from a suppressor hybridoma function via an antigen bridge at the TH or at the TH-dependent B-cell subset.

Analysis of mammalian scrapie protein by novel monoclonal antibodies recognizing distinct prion protein glycoforms: an immunoblot and immunohistochemical study at the light and electron microscopic levels.

The availability of specific monoclonal antibodies (mAbs) recognizing the aberrant form (PrP(Sc)) of the cellular prion protein (PrP(C)) in different mammalian species is important for molecular diagnostics, PrP(Sc) typing and future immunotherapy. We obtained a panel of anti-PrP monoclonal antibodies in PrP(0/0) knock-out mice immunized with recombinant human PrP(23-231). Two mAbs, recognizing PrP epitopes in the alpha-helix 1 (mAb SA65) and alpha-helix 2 (mAb SA21) regions, immunoreacted with PrP(C) and PrP(Sc) and its proteolytic product, PrP27-30, from human, murine, bovine, caprine and ovine brains by Western blot. Remarkably, mAb SA21 recognized unglycosylated and monoglycosylated PrP with the second site occupied by glycan moieties, but not monoglycosylated PrP with the first consensus site occupied or highly glycosylated species. Immunoblots with mAb SA21 disclosed that PrP glycosylated at the second site accounted for the slower migrating form of the customary monoglycosylated PrP doublet. mAb SA65 immunolabelled all PrP glycoforms by Western blot and was highly efficient in detecting tissue PrP by immunohistochemistry in light microscopy and in immunoelectron microscopy. These novel anti-PrP mAbs provide tools to investigate the subcellular site of PrP deposition in mammalian prion diseases and may also contribute to assess the role of different PrP glycoforms in human and animal prion diseases.

Effects of dietary wheat germ deprivation on the immune system in Wistar rats: a pilot study

Bioactive molecules that can gain access to body tissues through the gastrointestinal tract may interact with immune regulatory circuits and effector functions. Among these are plant lectins, such as wheat germ (WG) agglutinin, which constitute common components of the human diet and target the immune system on a daily basis. Dietary bioactive molecules might be considered as immunomodulatory signals. To investigate the possible effects on the immune system of the long-term absence of such signals, two groups of rats were fed on a diet containing or deprived of WG. The WG-deprived diet induced a state of functional unresponsiveness in lymphocytes from primary and secondary lymphoid organs, as evaluated by in vitro stimulation with T cell mitogen phytohemoagglutinin (PHA) and B cell mitogen lypopolysaccarides (LPS). The unresponsive state of the immune cells could be reversed by injection of antigen emulsified in oil with inactivated mycobacteria (complete Freunds adjuvant, CFA) Dietary signals can thus interact with the immune system possibly influencing its shaping during ontogenesis.

Induction of an antitumour adaptive immune response elicited by tumour cells expressing de novo B7‐1 mainly depends on the anatomical site of their delivery: the dose applied regulates the expansion of the response

De novo expression of costimulatory molecules in tumours generally increases their immunogenicity, but does not always induce a protective response against the parental tumour. This issue was addressed in the mouse Sp6 hybridoma model, comparing different immunization routes (subcutaneous, intraperitoneal and intravenous) and doses (0·5 × 106 and 5 × 106 cells) of Sp6 cells expressing de novo B7‐1 (Sp6/B7). The results can be summarized as follows. First, de novo expression of B7‐1 rendered Sp6 immunogenic, as it significantly reduced the tumour incidence to ≤15% with all delivery routes and doses tested, whereas wild‐type Sp6 was invariably tumorigenic (100% tumour incidence). Second, long‐lasting protection against wild‐type Sp6 was mainly achieved when immunization with Sp6/B7 was subcutaneous: a dose of 0·5 × 106 Sp6/B7 cells elicited protection that was confined to sites in the same anatomical quarter as the immunizing injection. Repeated injections of the same dose extended protection against wild‐type Sp6 to other anatomical districts, as well as a single injection of a 10‐fold higher dose (5 × 106 cells). Finally, Sp6‐specific cytotoxic T‐lymphocyte activity was detected in draining lymph nodes, and the splenic expansion of Sp6‐specific cytotoxic T‐lymphocyte precursors quantitatively correlated with the dose of antigen. We conclude that activation of a protective immune response against Sp6 depends on the local environment where the immunogenic form of the ‘whole tumour cell antigen’ is delivered. The antigen dose regulates the anatomical extent of the protective response.

Specific and long-lasting suppression of rat adjuvant arthritis by low-dose Mycobacterium butyricum

We have tested the therapeutic effect of intraperitoneal injections of Mycobacterium butyricum on the development of adjuvant arthritis in rats and we have explored the specificity and the duration of effectivity of this treatment. Rats with induced arthritis were injected intraperitoneally with the causative antigen, Mycobacterium butyricum, at concentrations 10 times lower than the inducing one, on the 3rd and 10th day after arthritis induction. The severity of the disease was assessed on the basis of physical (arthritis index, paw swelling) and biochemical (serum interleukin-6) parameters. The treatment with Mycobacterium butyricum led to a significant suppression of adjuvant-induced arthritis. This therapeutic effect was both antigen-specific, because intraperitoneal aspecific inflammation did not prevent the disease, and long-lasting. The results obtained in this model confirm the possibility of modulating the autoimmune process even when the immunological response is already triggered, suggesting new therapeutic strategies, more suitable than preventive vaccination, in human autoimmune diseases.

Activation of idiotype-specific suppressor T cells by injection of antibody carrying a recurrent idiotype

Intravenous injection of spleen cells (SC) coated with an antitrinitrophenyl (anti-TNP) IgM monoclonal antibody, Sp6 (Sp6-SC), which carries a recurrent idiotype, resulted in activation of a Lyt-2-positive population which did adhere to Sp6-coated plates. No effect of Sp6-SC injection could be observed in vivo on an anti-TNP B-cell response when mice were primed with an immunogenic dose of TNP-horse red blood cells (HRBC), but an anti-TNP response was observed when Sp6-SC-injected mice were primed with a subimmunogenic dose of TNP-HRBC. Furthermore, after intravenous (iv) injection of Sp6-SC, it was no longer possible to suppress a primary anti-TNP response by iv injection of TNP-haptenized thymocytes. In vitro analysis showed that the Sp6-induced suppressor T cell (Ts) population had no measurable influence on TNP-specific naive B cells, nor did it suppress TNP-specific helper T cells (THTNP), but it did lead to counterregulation of TNP-specific suppressor T cells (TsTNP). Hence, iv injection of antibody carrying a recurrent idiotype resulted in activation of a Ts population which functioned as inhibitor of suppression, thus displaying a helper effect.

A non-parametric method for the analysis of experimental tumour growth data

Analysis of tumour growth is required to investigate the biology of tumours and to determine the effects of new anti-tumour therapies. A non-parametric mathematical method for the analysis of a set of experimental tumour growth data is described. The method is based on the similarity between time series of tumour size measurements (e.g. tumour volume), similarity being defined as the Euclidean distance between data measured for each tumour at the same time. Subsets of similar time series are found for a given population of tumours. A biologically meaningful parameter H has been derived which is a measure of the scattering of experimental volume samples. The method has been applied to the analysis of the growth of (i) untreated multicellular tumour spheroids obtained with different cell lines and (ii) spheroids, treated with cytotoxic drugs (immunotoxins). Results are compared with those previously obtained by applying the classical Gompertz growth model to the analysis of treated and untreated spheroids.