Development and characterization of a rat model for locally recurring mammary tumors: sensitivities to 5-fluoro-2'-deoxyuridine, adriamycin, and X-irradiation

Local recurrence occurs in 4-47% of breast cancer patients and is often associated with development of metastatic foci and resistant cell populations. Thus, recurrent breast cancer indicates a poor prognosis for the patient. Local tumor-derived 13762NF rat mammary adenocarcinoma cell clone MTF7(T20) was injected into the inguinal mammary fat pad and allowed to grow before surgical excision. Individual locally growing (primary) tumors were removed and established in short-term tissue culture. Corresponding local recurrences were excised after regrowth and established in short-term tissue culture. All sublines were tested for in vitro sensitivities to 5-fluoro-2'-deoxyuridine, Adriamycin, and ionizing X-irradiation. Using a clonogenic colony formation assay, responses of individual sublines ranged from 85 to 1500 ng/ml for Adriamycin and 65 to 10,000 nM for FdUrd. Some recurrences were significantly more resistant while others were more sensitive than the corresponding primary tumor lines. All recurrences had smaller 90% lethal dose values than the corresponding parent or primary tumor in response to Adriamycin; whereas, to 5-fluoro-2'-deoxyuridine, 90% lethal dose values revealed that most lines were quite resistant. Statistically significant differences in radiation survival were observed only for lines LR1a and LR5 (more sensitive). There was no apparent correlation between sensitivities to chemotherapy agents or X-irradiation and experimental metastatic potential in LR sublines. These dose-response data indicate that locally recurrent tumors are frequently, but not always, different from the original primary tumor in response to chemotherapy agents and ionizing X-irradiation. Although an exact mechanism is unknown, it is likely that "selective" pressures which eliminate large numbers of cells, in this case surgery, change tumor composition so that recurrent tumors may no longer be equivalent to the tumor mass that was originally excised. This suggests that treatment strategies should be planned accordingly.     

Granulomatous mastitis: a report of seven cases

The clinical history and histological features of seven cases of granulomatous mastitis are presented. The lesion occurs in young parous women as a tender extra-areolar breast lump. Histologically, non-caseating discrete granulomas are present, confined to breast lobules with, in three cases, coalescence of the granulomas and microabscess formation. Pathogenesis of the changes is discussed. It is thought that granulomatous mastitis is an entity morphologically distinct from duct ectasia/plasma cell mastitis and the commoner forms of granulomatous breast diseases.     

Mitogenic activities of amino acid substitution mutants of staphylococcal enterotoxin B in human and mouse lymphocyte cultures.

Site-directed mutagenesis has been used to introduce amino acid substitutions at specific residues of the staphylococcal enterotoxin B (SEB) gene cloned from Staphylococcus aureus 10-275. The mitogenic activities of these derivatives were determined in two assay systems: (i) mouse spleen cells and (ii) a mixture of human peripheral blood mononuclear cells and lymphocytes. Substitution of either His-12, His-32, His-121, His-166, Lys-152, or Gly-205 did not significantly alter the mitogenic activity from that of the wild-type toxin in either proliferation assay. Substitution of either residue Asn-23, Phe-44, or Cys-93 reduced the mitogenicity of SEB by a degree that depended upon the assay system used. Similar to the results reported by others measuring toxin activation of mouse lymphoid cells, we found that substitutions of these three residues of SEB caused at least 800-fold reductions of mitogenic activity from that of the wild-type toxin. When tested for toxicity in vivo in D-galactosamine-treated mice, the reduced activities of these mutant toxins, however, were not as pronounced. In contrast, when tested in the human cell mitogenicity assay, these mutant toxins were active. Small alterations in activity (two- to fivefold reduction) were observable only at low concentrations. Our findings reveal the importance of using human lymphocytes in addition to the traditional mouse spleen cell assay when assessing biological activities of staphylococcal enterotoxins.     

Five year study of prenatal testing for Huntington''s disease: demand, attitudes, and psychological assessment.

Adult predictive and prenatal testing programmes for Huntington's disease (HD) in Canada have been available since 1986. However, the demand for prenatal testing and the reasons why some people choose not to have the prenatal test for this late onset disorder have not been well documented. In addition, the knowledge and attitudes of adult predictive testing candidates and their partners about prenatal testing are not well known nor are the psychological effects of prenatal testing well understood. As of September 1991, 425 subjects had entered the Canadian Collaborative Study of Predictive Testing and, of these, 47 subjects or their partners had become pregnant. Of this group, 14 (30%) couples requested prenatal testing, 24 (51%) couples did not want prenatal testing, and nine (19%) at risk subjects had already received a decreased risk through adult predictive testing and, therefore, were not eligible for the prenatal test. Of the 14 couples who initially requested prenatal testing, seven withdrew. Thus, demand for the prenatal test by eligible candidates was 7/38 or 18%, which is much lower than the 32 to 65% expected based on early survey data. The most frequently cited reason for declining prenatal testing was the hope that a cure would be found in time for their children. While the majority of adult predictive testing candidates (71%) in our study had accurate information about definitive prenatal testing, many (63%) did not have a correct understanding of exclusion prenatal testing. Although no serious adverse events such as suicide planning or admission to psychiatric hospital have occurred, a particular need for careful counselling was identified for those at risk candidates and their partners who have one prenatal test and feel compelled to use the test again in future pregnancies. Even though prenatal testing for HD is not requested as often originally expected, it still remains a desired option for some at risk persons and their partners.     

Selective and differential medium for recovery of Pseudomonas cepacia from the respiratory tracts of patients with cystic fibrosis.

A selective and differential medium, OFPBL (oxidation-fermentation base supplemented with agar, lactose, and two antimicrobial agents), for the isolation of Pseudomonas cepacia from respiratory specimens of patients with cystic fibrosis was developed and tested. Among 725 specimens submitted from seven centers over a 4- to 6-month period, 58 (8%) yielded P. cepacia on OFPBL; only 19 of these were recovered on MacConkey or sheep blood agar (P less than 0.001). No isolate was recovered on MacConkey or sheep blood agar alone. Ranges of recovery rates among centers were 0 to 15% on OFPBL and 0 to 10% on MacConkey or sheep blood agar. Ninety percent of P. cepacia isolates were detected on OFPBL in less than or equal to 3 days. Other nonfermenters and yeasts isolated on OFPBL were distinguished from P. cepacia by failure to acidify the medium. The new medium was clearly superior to MacConkey and sheep blood agars for the isolation of P. cepacia from the respiratory secretions of patients with cystic fibrosis.     

Rochalimaea henselae sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchymal bacillary peliosis.

Nine strains of Rochalimaea spp. that were isolated from patients over a period of 4.5 years were characterized for their enzyme activities, cellular fatty acid compositions, and DNA interrelatedness among Rochalimaea spp., Bartonella bacilliformis, and Afipia felis (cat scratch disease bacillus). All except one isolate, which was Rochalimaea quintana, were determined to belong to a newly proposed species, Rochalimaea henselae sp. nov. After recovery from clinical material, colonies required 5 to 15 days of incubation to become apparent. Cells were small, gram-negative, curved bacilli and displayed twitching motility. Enzyme specificities for amino acid and carbohydrate substrates showed that R. henselae could be distinguished from Rochalimaea vinsonii by L-arginyl-L-arginine and L-lysyl-L-alanine peptidases, but not all strains could be distinguished from R. quintana on the basis of peptidases or carbohydrate utilization. R. henselae also closely resembled R. quintana in cellular fatty acid composition, with both consisting mainly of C18:1, C18:0, and C16:0 fatty acids. However, the strains of R. henselae all contained C18:0 in amounts averaging greater than or equal to 22%, in contrast to R. quintana, which contained this cellular fatty acid in amounts averaging 16 and 18%. DNA hybridization confirmed the identification of one clinical isolate as R. quintana and showed a close interrelatedness (92 to 100%) among the other strains. Under optimal conditions for DNA reassociation, R. henselae showed approximately 70% relatedness to R. quintana and approximately 60% relatedness to R. vinsonii. Relatedness with DNA from B. baciliformis was 43%. R. henselae was unrelated to A. felis. R. henselae is the proposed species of a newly recognized member of the family Rickettsiaceae, which is a pathogen that may be encountered in immunocompromised or immunocompetent patients. Prolonged fever with bacteremia or vascular proliferative lesions are clinical manifestations of the agent.     

Multiple phenotypic divergence of mammary adenocarcinoma cell clones.

We have shown that, with in vitro passage, subclones derived from clonal cell populations of 13762NF mammary adenocarcinoma undergo phenotypic drift and diversification in their cellular properties. Here we examine whether phenotypic divergence of 13762NF cell clones extends to therapeutic treatments used in eliminating mammary tumors and whether the apparent rates of phenotypic divergence vary for different treatments. Six subclones isolated from low passage clone MTF7 (T11 l; tissue culture passage 11) cells were compared to a similar number of subclones isolated from high passage clone MTF7 (T35; tissue culture passage 35) cells. Subclones derived from clone MTF7 (TI l) were relatively homogeneous (not significantly different) in their inherent sensitivities to ionizing radiation, extrapolation coefficients and quasithreshold dose values (D_o = 1·61–1·99 Gy; n=0·89–3·42; D_q = 0–2·34). When the MTF7 (Tll) subclones were examined for their sensitivities to 45°C hyperthermic treatment, the inherent sensitivities and dose-response curve parameters (D_o = 5·24–10·05 min; n = 1·08–10·47; D_q = 0·78–12·31) were heterogeneous (significantly different). In addition, the MTF7 (T1 l) subclones were heterogeneous (significantly different) in their sensitivities and dose-response curve parameters to 5-fluoro-2-deoxyuridine (FUdR) treatment (slope= –0·70 to –1·59; y-intercept = 1·31 × 10² to 47·80 × 10²). The LD₅₀ values for FUdR ranged from 14–150 nm for the MTF7 (T11) subclones. At high passage MTF7 (T35) subclones were heterogeneous in their dose-response parameters to ionizing radiation (D_o = 1·17–2·05 Gy; n = 0·80–41·18; D_q = 1·79-\24·94), hyperthermia (D_o = 3·57–6·32 min; n = 2·08–13·54; D_q = 3·68–9·30) and FUdR (slope= –0·77 to –0·93; y-intercept = 4·64 × 10² to 8·83 × 10²; LD₅₀ = 50–160nm). The results indicate that clonal cells diverge for distinct phenotypic properties at differing rates to form heterogeneous cell populations with unique sensitivities to various therapeutic treatments.     

Idiotypic relatedness of human monoclonal IgG cryoglobulins.

The serological relatedness of the idiotypic (ID) determinants of one type lambda light chain dimer and fifteen monoclonal IgG cryoglobulins were assayed. Rabbits were immunized with 9/15 IgG cryoglobulins, and twenty-three antisera were obtained and absorbed to render them specific for the ID determinant of the immunizing IgG cryoglobulin. By use of haemagglutination-inhibition, cross-reactivity was detected among five cryoglobulins. This was localized to the Fab region of the IgG, was not related to identity of the variable region subgroups of the heavy and light chains of the cross-reactive cryoglobulins, and was not detected in eighteen non-cryoprecipitable IgG myeloma proteins. The serological relatedness of the ID determinants suggests that a subset of IgG cryoglobulins may possibly have similar variable region structures and/or antigenic specificities.     

Increased frequency of rheumatoid factor precursor B lymphocytes after immunization of normal adults with tetanus toxoid.

Vaccination of normal adults with tetanus toxoid induced a two-three-fold rise in the frequency of IgM anti-IgG (rheumatoid factor, RF) B lymphocytes inducible by the polyclonal B cell activator, Epstein-Barr virus. The increase in IgM-RF precursors occurred earlier, was greater in magnitude, and was more sustained than the change in plasma IgM-RF. It was associated with a rise in total IgM levels, and correlated positively with the magnitude of the IgG anti-tetanus antibody response, but not with levels of circulating immune complexes. The ability of apparently innocuous infections and immunizations to increase the frequency of IgM-RF precursor B lymphocytes may be the reason for the previously noted expansion in this autoreactive B cell pool between birth and adulthood.