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BIOASSAY- CARCINOGENFSIS & TISSUE CULTURE THL COUNCIL FOR TOBACCO RESLARCIi - U.S.A. 633 TIIl[2D AVBNIIE NEW YOIiK, N. Y. 10017 CO'iMITTF.,E : Dr. Bing, Chm. Dr. Sommers Dr. Reimann Application For Renewal of Research Grant First Z] Second Q 1. Name of lnvestigator(s): (include title and degrees) Geoffrey L. Brinkman, M.D., Associate Professor of Medicine Wayne State University School of Medicine 1400 Chrysler Freeway Detroit, Michigan 48207 - : 3. Short Title of Projects The Effect of Cigarette Smoking on the Ultramicroscopic Structure of the Bronchial Mucosa 4. Proposed Renewal Starting Date: (Anniversary or other) December 1, 1967 5. Discuss any Important Changes or Additions to Objectives or Specific Aims: • Recently in the bronchial mucosa from an adult, non-smoking patient, some -structures have been seen which have the typicaL morphological appearance of tumor whether these virus-like bodies can be identified in other individuals, both in the bronchial mucosa and in lung tumors. For this reason, I plan to broaden the scope `of the study to include study of the ultramicroscopic structure of lung cancers. If ,neoplasms cannot be.decided at this time. However, further study is indicated to see ; particular biopsy. Whether they are oncogenic or in any way related to bronchogenic virus (Figures 1 & 2). These virus-like bodies were seen in several cells from this '-~..:-these virus-like bodies can be recognized in lung tumors, then further study will be ,justified to determine whether they have oncogenic properties. Representative photomicrographs of some recent biopsies are attached (Figures 3-10). Up until now it has been impossible to obtain bronchial biopsies in normal individuals and we have had to rely on patients who are undergoing lung resection and in whom there was no clinical~or pathological evidence of bronchial mucosal ab- normality. I have now been able to make arrangements with Children's Hospital of Michigan to obtain bronchial biopsies from children undergoing lung resection for removal of congenital pulmonary abnormalities, and as a result we will be able to ~A obtain bronchial tissue which probably can be regarded as normal. C 0 C031 Ul ~ ~ ~ ~ J 6. Give a Brief Statement of your Working Hypothesis if altered or modifieda ~. ..~.~:.A........,~..:.:~.. .__. ~_.._.._ ~. ._._.,..~ :,:,

. Changes or Additions to Experimental Design and Procedures: (Attach Separate Pages). No basic change in experimental design or procedure is planned, but we have ;`enable us to determine what is the best procedure for processing human bronchiaL these variations of technique are proceeding and ultimately will, I am sure, embedding material (Vestophal and Maraglass). Experience with comparison of -experimented during the year with different strength fixatives and different mucosa. 9 9. Changes in Personnel with Biographical Sketches of new Personnel (append)'. My original technician is now working on a part-time basis because of domestic problems, but as well, I have another part-time technician who is an undergraduate student in Biology at Wayne State University. Between the two technicians I have the equivalent of a full-time technician. *0. Publications or Papers in Press resulting from the Project or closely related work

71 rA W rJL PrI {9 t: Technical ,Mary Ann Perczak Social Security C. Other Expenses (itemize) .Service Contract D. Permanent Equipment (itemize) Viewing binoculars E. Overhead (15% of A+ B-FC) 6 w : -.., ~ , . , , .. .. .. . _ . . . . _.. .. } A. Salaries (Personnel by names or category) Professional Geoffrey L. Brinkman B. Consumable Supplies(list by categories) :Photographic Chemicals, embedding materials Glassware }1. Budget (for coming year) t~SL ~ E - ^-4_~`• Total Sub-Total. Sub-Total I Sub-Total 100 w%C W*J dd .. _. . . ; ... . .. . ~ ~ ' . ~,.k ., , % time Amount 33-1/3 Nil 7,000. 290. 7,290. 750'. 500. 100. 1,350. 1,400. 1,400. 563.' 1,506, 12.109. 5Y It is understood that the applicant and institutional officers in applying for a grant have read and found acceptable the Councills "Statement of Policy Containing Conditions and Terms Under Which Projecf Grants Are Made." / Signature ~"/. Dive o~ of~Pruj..ct .~, . -7% /Telephone Signature Of/i<.r of tM. InstltuNon.

w ® List finaeial support for research fro m all sources, including own institutioa for this and/or related research projects. \ I :, . I c ~= Other Sources of Financial Support Rand Foundation Source Detroit TB & Health Society American Cancer Society II Q Amount $39,000. $20,795. $11,847. '~F' ~ ~~.J. lw}I Duration LB; , w d a 3 2 , , Current Gift to purchase electron microscope'and to equip darkroom. Effect of Cigarette Smoking on Ultramicroscop Structure of the Human Bronchial Mucosa f

2T49DSCOOt Cr = cilia P = pellicular structures = lumen = "whiskers" Dg = degenerating cell = cell membrane = microvilli = intercellular spaces = vacuoles = mitochondria = cisternae Tf = tonofibrils D = nucleus = desmosomes R = ribrosomes Sc = scrolls G = granule

M49DSE001 FIGURE 1. A cell in the bronchial mucosa showing large cisternae which probably represent Golgi organ. In the surrounding cytoplasm are numerous vacuoles, some of which contain virus-like bodies consisting i n of a central core with a surrounding two or three-layered concentric wall. These are better, seen the next figure.

FIGURE 3. This photomicrograph was obtained from a man who was a heavy smoker and shows the luminal surface of a ciliated cell with several cilia which appear normal and a great deal of pellicular material between the cilia. This pellicular material is a branching form of particularly large microvilli and greatly increases the specific surface area of the cells. On the right hand side there is a dark cell which is an exhausted goblet cell. The microvilli on this cell are covered with "whiskers", which are seen in greater detail in Figure 4.

( FIGURE 4. Shows the microvilli with "whisker" formation. This has been noted frequently on cells which appear to be exhausted and about to be shed into the bronchial lumen. On the left, the pellicular material is seen in greater detail. .

i FIGURE 5. This is a cross-section of the tracheal mucosa and shows cells in the process of being shed into the tracheal lumen. The cell nearest the lumen on the right has a light cytoplasm and very little intracellular structure. The intercellular processes which are normally interlocking are well seen.

FIGURE 6. This was also obtained from the tracheal mucosa, but shows a cell in the process of being expelled into the lumen.

FIGLTRE 7. Shows two cells adjacent to the bronchial lumen. The one on the rijht is a normal ciliated cell with~~~~eil-formed cilia and pellicular material containin~ laroe numbers of mitochondria which extend up close to the basal body of the cilia. • The cell on the left has a nude appearance due to the loss of the luminal structures. The mitochondria in this cell are much sparser, giving the cell an empty appearance. This cell is probably in~a state of exhaustion and about to be shed into the lumen.

FIGURE 8. Shows tracheal mucosal cells which were obtained adjacent to a squamous cell carcinoma of the tracliea. This figure shows well-developed desmosomes, while in the cells themselves there are numerous tonofibrils which may indicate that this cell is reverting to a more primitive state. sM9VSEOOZ

FICURF 9. The next four photomicrographs were obtained from grossly normal bronchial mucosa in a paticnt witli squamous cell carcinoma. This figure shows a large cell containing numerous vacuoles with varyin~; types o' substructure within. As well, large cisternae are noted. The cell on the right of this figure shows well-developed tonofibrils.

FIGURI: 10. Shows the granules seen in the previous figure two and three-layered membranes are forming, some of which of the virus-like bodies seen in Figures 1 and 2. in greater detaii. Within these granules are concentric and begin to have the appearance

FIGURE 11. Shows these granules at greater magnification with these unusual scroll-like forming within the granules. zV4stscooi substructures

FIGURF 12. Further demonstration of these scroll-like substructures, some of which bear close re- semhlance to the virus-like bodies. The possibility that this cell is the site of either multiplication or destruction of viruses must be entertained. EV49VSEOOZ