Threshold concentrations of Streptococcus pneumoniae type 3, Haemophilus influenzae type b, and Streptococcus sp. group B type Ib required for positive counterimmunoelectrophoresis reactions were determined in vivo and in vitro. Animals were infected intraperitoneally with various concentrations of microorganisms: adult mice with S. pneumoniae, suckling rats with H. influenzae, and 3-week-old mice with Streptococcus sp. group B. At 24 h after infection a minimum blood concentration of 10(3) colony-forming units (CFU)/ml was needed for S. pneumoniae or H. influenzae before antigen was detected in the serum. A minimum concentration of 10(6) CFU/ml was needed for Streptococcus sp. group B at 10 h after infection. Larger threshold concentrations (10(4) CFU/ml for S. pneumoniae, 10(5) CFU/ml for H. influenzae, and 10(7) CFU/ml for Streptococcus) were required in broth-grown cultures before cell-free antigens could be demonstrated by counterimmunoelectrophoresis in the medium. Marked levels of antigen release by group B streptococci were observed as the cultures entered early stationary phase. This study provides evidence of a long-accepted, though poorly substantiated, hypothesis that a threshold concentration of microorganism is necessary before counterimmunoelectrophoresis reactions become positive. Counterimmunoelecrophoresis results for clinical specimens should be interpreted cautiously in light of this evidence.

A Cytospin slide centrifuge was used to concentrate 0.05- to 0.5-ml samples of cerebrospinal and other body fluids for Gram stain. Trials with cerebrospinal fluid containing known numbers of microorganisms indicated that the Cytospin increased the sensitivity of cerebrospinal fluid Gram stains by up to 2 logs compared with unconcentrated and conventional centrifuge smears. Cytospin-concentrated smears were prospectively compared with unconcentrated Gram-stained smears and bacteriological culture results for 80 clinical body fluid specimens. Bacteria were seen in unconcentrated smears of 9 of the 16 (56%) fluids which were infected, whereas Cytospin smears of 12 of the 16 (75%) showed bacteria. Cytospin smears revealed more bacteria and demonstrated better leukocyte morphology than did unconcentrated or conventionally centrifuged samples of small volumes of infected body fluids, allowing early diagnosis of infection.

Forty-eight strains of Streptococcus pneumoniae were tested in vitro to determine the minimum number required for pneumococcal capsular antigen to be detectable by latex agglutination. It was found that 10(6) to 10(7) microorganisms per ml were needed and that antigen remained detectable even when viable pneumococci could no longer be demonstrated.

We have studied RB protein expression in 171 cell lines derived from patients with small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), pulmonary carcinoid, mesothelioma, and extrapulmonary small cell cancer (EPSC) and have correlated this data with clinical outcome. We detected absent or aberrant RB protein expression in 66/75 SCLC, 12/80 NSCLC, 1/6 carcinoid, 0/5 mesothelioma, and 4/5 EPSC samples. In addition, we observed integration of human papilloma virus (HPV) DNA in the single EPSC cell line that retained wildtype RB protein. We did not detect integration of HPV, SV40 or adenoviral DNA in other tumor samples with wildtype RB status. We also noted a stable, hypophosphorylated mutant RB in 12 SCLC and 3 NSCLC samples which might have been falsely interpreted as wildtype by current immunohistochemical techniques. Analysis of the matched clinical data showed no associations between RB status and age, sex, extent of disease, performance status, smoking history, and previous treatment. In addition, retrospective analyses showed no consistent correlation of RB protein expression with either best clinical response, overall survival, or in vitro chemotherapeutic drug sensitivity. The stable expression of RB after gene transfection into RB(-) SCLC cells, however, resulted in a trend toward increased in vitro resistance to etoposide, cisplatin and doxorubicin.

The antigen expression of human small cell lung cancer (SCLC) was studied using a panel of 21 independent rat monoclonal antibodies. The panel was selected by isolating hybridomas producing antibodies reactive with two SCLC lines but not with autologous B-lymphoblastoid lines. The antibodies were then tested in radiobinding assays against a panel of 17 SCLC lines, 13 non-small cell lung cancer lines, 6 SCLC necropsy specimens, 13 neuroectodermal lines (melanomas, neuroblastomas, glioblastomas), 15 other human lines, the glycolipid extracts of SCLC, human meconium, and human red blood cells. Using immunohistochemical assays, 14 of the antibodies were tested against normal lung, liver, and kidney, and lung cancer biopsies and xenografts. These analyses revealed the following:(a) SCLC elicited predominantly immunoglobulin M antibodies despite hyperimmunization;(b) the 21 antibodies displayed distinct binding and immunohistochemical phenotypes, indicating that they recognized many different epitopes;(c) 14 of the 21 antibodies reacted with glycolipid determinants;(d) the 21 determinants were expressed on over 80% of SCLC cell lines, necropsy samples, and xenografts;(e) the determinants were also expressed on normal adult bronchial epithelium, proximal tubules of adult kidney, and in a few instances on other normal cell types;(f) the antigens were expressed less frequently on nonsmall cell lung cancer samples but did not clearly distinguish SCLC from non-small cell lung cancer;(g) biochemical and morphological variants of SCLC exhibiting more malignant and undifferentiated behavior and containing greatly amplified c-myconcogenes failed to express several determinants or expressed them at lower levels;(h) and finally, while many human cell lines failed to express the antigens including human melanoma and glioblastoma lines, human neuroblastoma lines frequently did express the SCLC antigens. These detailed studies utilizing a panel of distinct monoclonal antibodies define a series of antigens on the surface of the majority of SCLC undescribed previously.

A monoclonal antibody against purified calf DNA polymerase alpha (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) was used to immunoprecipitate proteins from a crude soluble extract of growing monkey BSC-1 cells. Immunoprecipitates contained familiar DNA polymerase alpha catalytic polypeptides of Mrs approximately equal to 115,000 and 70,000 and also a Mr 40,000 catalytic polypeptide; the major component in the immunoprecipitates, however, was a polypeptide of Mr approximately equal to 190,000 not previously identified as a DNA polymerase. This protein was capable of DNA polymerase activity after electroelution from NaDodSO4/polyacrylamide gels and renaturation. The highly purified enzyme so obtained was active with poly(dT).oligo(rA) as template.primer, resistant to dideoxy TTP (ddTTP), and inhibited by aphidicolin and butylphenyldeoxyguanosine 5'-triphosphate, thus identifying it as a DNA polymerase alpha. The results indicate that a polypeptide of Mr approximately equal to 190,000 is an abundant component among DNA polymerase alpha catalytic polypeptides in growing monkey cells.

Two murine IgG2Ak monoclonal antibodies (703D4, 704A 1) were produced and characterized after immunization with a human large cell lung cancer line (NCI-H 157). These antibodies detect different epitopes on 31 kilodalton [35S]methionine incorporating protein(s). Radiobinding and immunohistochemical studies show these antibodies bind to most (11/13) human non-small cell lung cancer (adenocarcinoma, epidermoid, and large cell), but not to small cell lung cancer (0/11) tumors tested. The epitopes these antibodies recognized are also expressed on human melanomas (7/8), two other tumors (osteogenic sarcoma, renal cell carcinoma), but not on many other human tumors (breast, colon, neuroblastoma, lymphoid), and not on a panel of normal adult human tissues. Because the antigen(s) are preserved after fixation and because of their ability to distinguish lung cancer types from each other and normal tissues, they should be of clinical, as well as of biologic interest.