HLA-DQB1 typing of north east Italian IDDM patients using amplified DNA, oligonucleotide probes and a rapid DNA-enzyme immunoassay (DEIA).

We report on HLA-DQB1 typing in IDDM patients of north east Italian region using an enzymatic method based on the detection of hybridization reaction between PCR amplified DNA from whole blood and allele specific oligonucleotides by an antibody directed against double stranded DNA (DNA-enzyme immunoassay or DEIA). The method is reliable, simple and sensitive as the classical radioactive method with the advantage of using a universal non radioactive detection reagent. Nineteen families, each including one subject with juvenile insulin-dependent diabetes mellitus (IDDM) were analyzed. A strong association between absence of an aspartic acid (Asp) in position 57 of DQB1 beta chain in homozygous conditions and susceptibility to IDDM was found. In contrast with some previous observations, however, no significant association was found between Asp/non-Asp heterozygous genotype and IDDM. No patients were found with an homozygous Asp/Asp genotype, known to be protective in caucasoid population. Of particular interest was the DQB1 allelic distribution in our population sample. The non-Asp allele most frequently found in IDDM subjects was the DQB1 0201 allele and this finding was statistically significant (Pc value < 0.05, relative risk = 5.01). No significant association was found for any other allele including the DQB1 0302 (Pc value = not significant although with relative risk = 3.28) previously reported as the most frequent allele in IDDM caucasoid patients.

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/f(delta) 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.

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 cloned cells if compared to 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.

A rapid and improved method for generating cDNA libraries in plasmid and phage lambda vectors.

We have developed a fast and efficient procedure for generating cDNA libraries in plasmid or phage lambda vectors. We used Mo-MuLV reverse transcriptase to synthesize the first strand and directly added Escherichia coli DNA polymerase I with RNase H to synthesize the second strand. A special advantage of our procedure is the use of oligodeoxynucleotide adapters to insert the cDNA into the vector, avoiding the use of methylating enzymes and subsequent digestion with massive amounts of restriction endonucleases. This also obviates the need to tail the cDNA molecules with homopolymers, simplifying subsequent procedures such as sequencing or transfer to other vectors. Finally, we have used a rapid screening procedure to isolate full-length clones with oligodeoxynucleotide probes recognizing conserved regions at the 5' termini of the mRNA. The system is ideal for cloning and analyzing polymorphic alleles of genes, such as those of the major histocompatibility complex.

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.

Cell contact-dependent PMN HLA-DR and CD69 membrane expression induced by autologous mono-lymphocytes and cell lines.

Polymorphonuclear granulocytes (PMN) are commonly considered short-lived cells playing an efficient role in primary host defense via phagocytosis and release of cytotoxic compounds and inflammatory cytokines. Purified PMN do not express HLA-DR and CD69 molecules on cell surface, but they can be induced to do so by co-culture with peripheral blood derived mono-lymphocytes. De novo cell-surface expression of HLA-DR was also induced in PMN by co-culture with cell lines of lymphoid phenotype, but not with cell lines of myeloid phenotype. CD69 expression was not induced by co-culture with any of the cell lines used in the present study. In addition, we have observed induction of HLA-DR surface expression on PMN by culture in presence of culture supernatant of one of the cell lines of lymphoid origin, RPMI-8866. Quantitative analysis of HLA-DR and CD69 expression in stimulated PMN allowed us to divide PMN donors in two main groups, one with low expression and the other with high expression of the two molecules. HLA-DR surface expression was not altered by treatment with CHX and BFA, and RT-PCR analysis of total RNA from resting and stimulated PMN with RPMI-8866 supernatant did not detect the presence of any specific HLA-DR and CIITA transcript. Flow-cytometry and fluorescence microscopy analysis of resting PMN revealed the presence of HLA-DR molecules localized in intracellular vesicular-tubular structures. These data show that a reservoir of HLA-DR molecules is stored in the cytoplasm of human resting PMN and can be released to reach cell surface by a mobilization mechanism induced by cell surface interactions with selected cell types and sometimes with molecules released in culture supernatants.

Polichemotherapy of advanced head and neck malignancies.

The favorable results obtained by other authors with polichemotherapy encouraged us to employ therapeutic scheme using a combination of 4 drugs. Treatment envolved the administration of 300 mg/mz cyclophosphamide, 350 mg/m2 5-fluorouracil, 10 mg/mw2 methotrexate i.v. on alternate days 6-8 times, and 15 mg bleomycin on alternate days until a total dose of 150-200 mg is reached. Thirty-five out of 37 patients treated with this protocol (30 previously treated and 5 not) qualified for analysis; the site of the neoplasm, mostly squamous cell carcinoma, was different; for the most part it was in the larynx (18/35) and the oral cavity (10/35). Complete remission was achieved in 9/35 patients (25.7%), varying from 5 to 33 months (median 22); partial remission was achieved in 15/35 cases (42.8%), varying from 1 to 14 months (median 3); and there was no success in 11/35 cases (31.5%). Overall, a total remission greater than 50% was observed in 24/35 patients (68.5%). The most serious side effects both ascribed to BLM were observed in the central nervous system (increasing drowsiness and coma) and the lung. This study has shown that in the ultra head and neck malignancies medical treatment can achieve satisfactory results.

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.

[Current status of chemotherapy of tumors of the head and neck]. / Stato attuale della chemioterapia nei tumori testa e collo.

The literature regarding chemotherapy of cervico-facial tumours is reviewed. Owing to its modest, short-lived results intra-arterial treatment has been largely abandoned even in the case of serious complications. It is only used in selected cases as a preparation for subsequent surgical and/or radiation therapy. General, one-drug cytostatic therapy has been given fair results with objective remissions varying from 7.5 to 58%. In recent years polychemotherapy has proved more effective with remissions of from 30 to 80%. There are too few data on the polychemotherapy-immunotherapy association to draw valid conclusions.

[Results of polychemotherapy in the advanced stages of cervico-facial tumors]. / Risultati della polichemioterapia negli stadi avanzati dei tumori del distretto cervico-faciale.

An association of cyclophosphamide, fluorouracil and methotrexate already employed with success against solid tumours in other sites was used in the treatment of 62 patients with advanced tumours of the head and neck. Complete (40.32%) and partial (43.55%) regressions were obtained relatively quickly. A satisfactory period of remission occurred in patients for whom no further treatment had seemed possible, either on account of the extent of the disease, or because surgery and/or radiotherapy had already been performed.