Coexpression of biological key modulators in primary colorectal carcinomas and related metastatic sites: implications for treatment with cetuximab.

BACKGROUND: Recent studies suggested substantial differences between primary tumors and metastases for EGFR expression in colorectal cancer (CRC). The aim of the study was to correlate the expression of a panel of molecular markers between primary CRC samples and metastases. METHODS: Expressions of EGFR, pEGFR, VEGF, pVEGF, PTEN, pAKT and p21 were analyzed in 28 primary tumors and 32 liver metastases by immunohistochemistry performed on formalin-fixed, paraffin-embedded sections from 46 CRC patients. The molecular profiles were evaluated by tissue micro-array. The correlation between tumor and metastasis biomarker expressions was tested. RESULTS: Among 60 CRC samples, 25% were EGFR positive, 38% were pEGFR positive, 38% were VEGF positive, 48% were pVEGF positive, 70% were pAKT positive and 51% were p21 positive. PTEN was deleted in 39% of cases and absence of p21 expression was found in 49% of cases. A significant correlation was observed between primary tumors and metastases for pAKT (p = 0.037) and pEGFR (p = 0.0002) status. In patients treated with cetuximab-based therapy (n = 18), p21 appeared as a significant predictive factor of response (p = 0.036). CONCLUSION: Biomarkers status may change between primary and metastatic sites in CRC, with potential implications for the identification of patients who are likely to respond to anti-EGFR treatment.

Influenza virus vaccine in B-cell chronic lymphocytic leukaemia patients.

The clinical reaction and the immunological response to influenza virus vaccine were studied in 43 B-cell chronic lymphocytic leukaemia patients. The Vaxigrip vaccine was administered containing the antigens A/Ghizhou/54/89, A/Singapore/6/86, and B/Yamagata/16/88. The side-effects observed were minimal and well tolerated. Antibody production with titres > 1:20 on day 15 was observed at least for one antigen in 35 patients (81%). In 23 of them (63%) this response was retained on days 30 and 60. Patients with IgG levels (< 700 mg/dl) responded less well as compared to those having normal IgG levels (> 700 mg/dl).

A nylon membrane enzyme immunoassay for rapid diagnosis of influenza A infection.

A new membrane-enzyme immunofiltration assay (MIFA) was developed for rapid diagnosis of influenza A infection. The pretreated specimens were dispensed into a 1.2 micron Biodyne B nylon membrane-bottomed microplate and vacuum filtration was applied. Blocking solution, peroxidase-conjugated anti-influenza A nucleoprotein monoclonal antibody, washing buffer and substrate were added in that order. The assay was completed within 30 min. Out of 103 nasopharyngeal swabs collected in transport medium, 31 isolates of influenza A virus were obtained and 22 specimens were detected directly by the MIFA technique. The 9 isolation-positive MIFA-negative specimens required 6 days or more for viral detection in cell culture, and probably contained a very low quantity of virus. The 72 cell culture negative specimens were also negative by MIFA. Comparison with a classical immunocapture assay (ICA) gave a better sensitivity for MIFA, as only 15/103 specimens were positive by ICA. MIFA is a rapid test with 71% sensitivity and 100% specificity. It was also very useful to test the cell culture supernatants, as a sensitivity of 100% was obtained with MIFA when the immunofluorescence technique was positive. The same technique could be readily carried out on the same plate for other respiratory viruses since capture antibody is not used.

Effects of iron deficiency upon the antibody response to influenza virus in rats.

The effects of severe and moderate iron deficiency upon the antibody response to influenza virus were investigated in rats. Three groups of weanling male Wistar rats were fed one of two iron-deficient diets (5 mg and 15 mg iron/kg diet) or a normal iron-containing diet (35 mg iron/kg diet). A group of individually pair-fed rats was introduced with the low iron-consuming rats. The effects of the diets upon various iron status parameters were followed during the 4th, 5th, 6th, and 7th week of diet. After 4 weeks of feeding different diets, an intraperitoneal injection of inactivated influenza virus A/New Jersey/76 was performed and a recall injection was done at 5 weeks. Primary and secondary antibody responses were assayed. Rats were sacrificed at 7 weeks of diet. After 4 weeks of feeding different diets, the rats fed the 5 mg iron/kg diet were severely anemic and rats fed 15 mg iron/kg diet were moderately iron-deficient, as shown by their iron status parameters. Growth was delayed in anemic and matched pair-fed rats. A primary antibody response was almost nonexistent in all groups. Secondary antibody titers were significantly weaker in anemic rats than in ad libitum controls, but were not different from those of pair-fed rats. This response was similar in moderately iron-deficient, ad libitum, and pair-fed rats. These results show that antibody synthesis in response to the influenza virus vaccine is preserved in moderate iron deficiency but is reduced in severe anemia. The reduction in energy consumption associated with severe iron deficiency in the rat could play a part in the altered humoral response.

In vitro human cytotoxic T cell responses against influenza A virus can be induced and selected by synthetic peptides.

Studies on human anti-influenza cytolytic activities have demonstrated that cytotoxic T lymphocytes (CTL) from HLA-B37 individuals react preferentially with the peptide corresponding to residues 335-349 of the nucleoprotein, whereas CTL from HLA-A2 donors recognize peptide 57-68 from the viral matrix as a dominant epitope. We studied the secondary CTL response, obtained from peripheral blood mononuclear cells, of an HLA-A2+,B37+ individual stimulated either by infectious virus or by synthetic peptides. Only an HLA-B37-restricted response was detected after stimulation by the whole virus, showing an immunodominance of this activity over that restricted by HLA-A2. Moreover, human cytotoxic cell lines were successfully obtained after stimulation of peripheral blood mononuclear cells with synthetic peptides. Under these conditions, it was possible to selectively reveal the existence of an HLA-A2-restricted activity directed against the matrix peptide. These results demonstrate that, at least in vitro, it is possible to stimulate a latent repertoire by using synthetic peptides. Nevertheless, we could not induce a response against the matrix or the nucleoprotein peptides in HLA-A2- or B37- individuals, suggesting that a finer selection of synthetic peptides would be necessary for their possible utilization to induce CTL during vaccination.

Fine specificity analysis of human influenza-specific cloned cell lines.

Influenza-specific human-T-cell clones, proliferating in the presence of virus-infected cells with restriction by class II molecules and displaying class II-restricted CTL activity or specific helper activity in antibody synthesis, have been analyzed for antigenic specificities. All of them were obtained by in vitro stimulation against influenza A/Texas virus. In all cases the virus specificity appeared identical in cytolytic and proliferative responses. Three of the clones were broadly cross-reactive, recognizing all or almost all type A influenza strains. The three remaining clones were subtype specific when tested with human strains and recognized the surface glycoproteins of influenza virus. One of these lines reacted with an epitope of the neuraminidase N2 while the other two recognized the hemagglutinin H3. By using a large panel of mammalian and avian influenza strains, it can be demonstrated that hemagglutinin-specific human T cells can recognize a cross-reacting determinant shared by H3 and H4 subtypes of hemagglutinin which has never been detected with antibodies.

Influenza A hemagglutinin-specific T cell clones strictly restricted by HLA-DR1 or HLA-DR7 molecules.

The antigenic specificities, major histocompatibility complex restrictions and functional properties of influenza virus-specific proliferative cloned cell lines have been studied. These lines were specific for the H3 hemagglutinin subtype of influenza A viruses. By using a large panel of HLA-phenotyped antigen-presenting cells, it was found that the polymorphic structures, defined as DR1 and DR7 molecules, or closely associated structures, function as the restricting elements. We excluded for these lines a possible restricting role of supertypic specificities, known cross-reacting elements on DR molecules, or products of other loci in known linkage disequilibrium with the HLA-DR molecules. Such exquisitely restricted clones might be of great help in the class II typing of antigen-presenting cells. Their specific activity was stable for several months. This has allowed the study of some functional properties of these long-term-cultured cloned cell lines: interleukin 2 sensitivity and production, helper function in specific antibody synthesis and ability to stimulate in mixed leukocyte reactions.

Bunyaviridae.

The family Bunyaviridae comprises over 200 viruses (serotypes, subtypes, and varieties) that infect vertebrates and/or invertebrates. Four genera of viruses have been defined (Bunyavirus, Nairovirus, Phlebovirus, and Uukuvirus). The main characteristics of the member viruses are: (i) the virus particles are for the most part uniformly spherical, 80-110 nm in diameter, and possess a unit membrane envelope from which protrude polypeptide spikes 5-10nm long; (ii) the viruses have three helical nucleocapsids, often in the form of supercoiled circles, each consisting of a single species of single-stranded RNA, major nucleocapsid polypeptide, N, and at least in some cases minor amounts of a large polypeptide which may be a transcriptase component; (iii) the genome is composed of three species of RNA (L, large; M, medium; and S, small), organized in end-hydrogen bonded circular structures; (iv) most viruses have three major virion polypeptides (N, and two surface polypeptides, designated G1 and G2); (v) for at least some member viruses, the virions have been shown to contain an RNA-directed RNA polymerase, believed to be responsible for the synthesis of viral complementary mRNA, so that bunyaviruses are considered to be negative-stranded viruses; (vi) at least some bunyaviruses are capable of heterologous virus genome segment reassortment and can form recombinant viruses at high or low frequency; (vii) viruses appear to mature primarily at smooth membrane surfaces and accumulate in Golgi vesicles and saccules, or nearby; (viii) transovarial, venereal and/or transstadial transmission in arthropods has been shown to occur for some members of the family.

Purification of haemagglutinin and neuraminidase from influenza virus strain 3QB and isolation of a peptide from an antigenic region of haemagglutinin.

Methods were developed for the purification of the surface, membrane-bound glycoproteins haemagglutinin and neuraminidase of influenza virus strain 3QB, in antigenically active forms. The methods employed in the purification included selective removal of the neuraminidase with the proteinase, bromelain, and subsequent disruption of the residual virus particle with the detergent Sarkosyl to release the haemagglutinin. Using techniques for proteolytic digestion of intact, native proteins an antigenically active peptide was isolated from the purified haemagglutinin, the surface glycoprotein against which the major antigenic response is directed. The amino acid composition of this peptide was determined. This was a 16-residue peptide with amino-terminal isoleucine and composition Ile1 Val1 Asx2 Thr1 Ser2 Glx2 Pro1 Gly3 Ala1 Leu1 Lys1.

Togaviridae.

The family Togaviridae is described; it contains four genera--Alphavirus, Flavivirus, Pestivirus and Rubivirus--and additional members. The main characteristics of the family are as follows: single-stranded, linear RNA, molecular weight about 4 X 10(6). Virions have isometric nucleocapsids surrounded by a lipoprotein envelope containing host cell lipid and virus-specified polypeptides including one or more glycopeptides. Virions yield infectious RNA. There are at least 80 members; all the alphaviruses and most flaviviruses are arboviruses in the biological sense.

Immune responses to influenza virus in rabbits after local immunization. II. Local and systemic cell mediated response.

The development of local and systemic cell mediated immunity (CMI) was investigated in rabbits after intratracheal immunization with live or inactivated influenza A virus. Lymphocytes from bronchoalveolar washings and draining lymph nodes were used for the assay of local CMI response. Spleen cells were employed for determination of systemic responses. Since alveolar macrophages were found to be susceptible to the action of migration inhibiting factor, the development of CMI in lungs was assayed by macrophage migration inhibition test using bronchoalveolar was cells. Immunization with live virus induces beter local CMI response than inactivated virus. After primary immunization the peak responses were found between second and third week. The secondary response was detectable much earlier and peaked at one week after booster. Small amounts of inactivates virus, which was unsufficient to evoke a primary response, could elicit a strong secondary response. The development of rapid and accentuated secondary response in the local lymphoid tissues suggests the presence of memory in the local CMI response. The results also show a good correlation to exist between primary local CMI response and the skin reactivity to the immunizing antigen. However, lack of such correlation during the secondary response leaves the skin tests less reliable in the evaluation of CMI in viral infections. Some of the functions of alveolar macrophages in the local immune reponses are also discussed.

Cellular aspects of local antibody response in rabbits.

Live or inactivated influenza virus is inoculated intratracheally to rabbit. After 4 days or more, lungs are washed with 50 ml of saline and fluid is examined for immunoglobulin and antibody content as well as for type and properties of free cells. IgA and IgG are detectable in the fluid but not IgM. Specific antiviral activity is associated only with IgA at the earliest stages. Free cells are so called 'alveolar macrophages' as demonstrated by shape and phagocytic or enzymatic properties. Their total numbers vary according to type of immunization: non-immunized 5.10-5. inactivated virus 2.10-6. live virus 10.10-6. A high percentage (50 to 90 percent) are positive in IgA IF test, only 10 percent with IgG test. Peroxydase electron microscopy shows intracellular presence of IgA.

Influenza virus and ciliary beating of the respiratory epithelium in sensitized animals.

Normally, under good survival conditions, the ciliary beating can be observed and recorded during hours after dissection of the respiratory epithelium. When laboratory animals have been immunized locally against influenza virus (under delayed type hypersensitivity conditions) the ciliary beating stops within 3 to 6 minutes when the same virus is put in contact with the sensitized mucosa. The specificity and the immunological conditions of this stopping have been analyed and the practical importance of this new test in cellular immunology is discussed.