The validity of self-reported dental agenesis.

The optimal tools for obtaining a diagnosis of dental agenesis (DA) among adults are childhood dental records and radiographs. However, these are often not available, and therefore the present study aimed to assess whether DA in adults could be validly assessed by self-reported information guided by a questionnaire. A questionnaire eliciting information on DA of permanent teeth was constructed, pilot-tested, and subsequently posted to a case group consisting of 334 young adults, for whom the DA status had been ascertained in the period from 1992 to 2002. A control group, consisting of 258 young adults randomly selected from the population from which the cases originated, was also approached with questionnaires. The response rate was 53.7% among cases and 46.4% among controls. The sensitivity and specificity of self-reported DA were estimated to be at least 0.88 (95% CI = 0.82-0.92) and 0.95 (95% CI = 0.89-0.98), respectively. These diagnostic test parameters are clearly inadequate for population-screening purposes. However, when screening patient groups with a higher occurrence of DA than is characteristic of the background population, the self-reported DA status may be a useful tool for identifying risk groups for conditions associated with the presence of DA.

Lung fibroblasts improve differentiation of rat type II cells in primary culture.

Epithelial-mesenchymal interactions mediate prenatal lung morphogenesis and differentiation, yet little is known about their effects in the adult. In this study we have examined the influence of cocultured lung fibroblasts on rat alveolar type II cell differentiation in primary culture. Type II cells that were co-cultured with lung fibroblasts showed significant increases in messenger RNA (mRNA) levels of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Metabolic labeling and immunohistochemistry demonstrated that these mRNAs were translated and processed. Addition of 10(-7) M dexamethasone (DEX) to cocultures antagonized the effects of the fibroblasts on SP-A and SP-C, but significantly augmented the effects on SP-B; expression of SP-D was unaffected. Coculture of type II cells with lung fibroblasts also increased acetate incorporation into phospholipids 10-fold, which was antagonized by DEX. Keratinocyte growth factor (KGF) mimicked the effects of lung fibroblasts on SP gene expression, but KGF neutralizing antibodies only partially reduced the effects of lung fibroblasts. KGF increased acetate incorporation into surfactant phospholipids, and the addition of DEX augmented this response. Together, our observations suggest that epithelial--mesenchymal interactions affect type II cell differentiation in the adult lung, and that these effects are partially mediated by KGF.

KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in adult rat lung.

Keratinocyte growth factor (KGF, FGF-7) is a potent mitogen for epithelial cells. We instilled recombinant human KGF to determine the effects of KGF on alveolar epithelial cells. Left lungs of adult rats were instilled intrabronchially with KGF (5 mg/kg) or normal saline. KGF instillation resulted in epithelial cell hyperplasia, and the alveolar bromodeoxyuridine (BrdU) labeling index peaked at 35% on day 2 after instillation. The mRNA levels for the surfactant proteins (SPs) SP-A, SP-B, and SP-D were increased in whole lung tissue on days 1 and 2 after KGF treatment and then returned to control levels on days 3-7. SP-C mRNA levels were increased on days 2-5 after KGF instillation. However, all surfactant protein mRNAs were reduced in type II cells isolated from rats instilled with KGF 2 or 3 days before isolation. These observations were confirmed by in situ hybridization. Instillation of KGF also increased the amount of SP-A and SP-D in lavage fluid. Transcripts for CC10, the 10-kDa Clara cell protein, were decreased. KGF increases the mRNA for the surfactant proteins per lung because of type II cell hyperplasia, but the mRNA per cell is slightly diminished as measured in isolated cells or estimated by in situ hybridization.

Effect of adenovector-mediated gene transfer of keratinocyte growth factor on the proliferation of alveolar type II cells in vitro and in vivo.

Alveolar type II cell proliferation occurs after lung injury and is thought to minimize the subsequent fibrotic response. Keratinocyte growth factor (KGF) has been shown to be a potent growth factor for rat alveolar type II cells. In this study, we created a replication-deficient, recombinant human type 5 adenovirus vector expressing human KGF (Ad5-KGF) to produce alveolar type II cell hyperplasia in vivo. In rat type II cells in vitro, Ad5-KGF at a multiplicity of infection (MOI) of 2, 4, and 8 plaque-forming units (PFU)/cell increased thymidine incorporation 13.3-, 16.8-, and 20. 8-fold, respectively. The KGF concentration in the medium increased up to 26.0 +/- 1.0 ng/ ml. We then instilled 10(9) PFU of Ad5-KGF, Ad5-LacZ, or phosphate-buffered saline into Fischer 344 rats and analyzed the lungs 2, 3, 7, 14, 21, and 28 d later. Ad5-KGF produced extensive alveolar type II cell hyperplasia on Days 2, 3, and 7. Surfactant protein (SP)-A and SP-D in lavage and SP-D in serum increased more in the Ad5-KGF group than in the Ad5-LacZ and PBS groups on Days 2 and 3. KGF was readily detectable for up to 7 d in lavage fluid, although only a modest number of cells expressed KGF messenger RNA as detected by in situ hybridization. These data show that Ad5-KGF stimulates extensive alveolar type II cell proliferation in vivo.

Surfactant protein C expression in urethane-induced murine pulmonary tumors.

Mice injected with urethane develop tumors with distinct histological patterns, which are classified as solid, papillary, or a mixture of these two patterns within the same tumor. Most investigators agree that solid tumors arise from alveolar type II cells, but the cellular origin of papillary tumors is less certain, being attributed to either type II cells or nonciliated bronchiolar epithelial (Clara) cells. To characterize the state of differentiation of these tumors more precisely and to provide additional information on gene expression, we used immunocytochemistry and/or in situ hybridization to determine the cellular localization of surfactant-associated proteins A (SP-A), SP-B, SP-C, and SP-D; Clara cell-associated protein CC-10; and thyroid transcription factor-1. In normal mouse lung, the messenger RNAs (mRNAs) for SP-A, SP-B, and SP-D were expressed in both type II cells and Clara cells. SP-C mRNA, however, was expressed only in type II cells, and CC-10 expression of mRNA was restricted to Clara cells. All tumors examined, both solid and papillary, expressed SP-A, SP-B, SP-C, SP-D, and thyroid transcription factor-1, but not CC-10. However, SP-C expression was slightly diminished in larger (older) papillary tumors. These results demonstrate that urethane-induced murine lung tumors express the type II cell phenotype.

Induction of alveolar type II cell differentiation in embryonic tracheal epithelium in mesenchyme-free culture.

We have previously shown that fetal lung mesenchyme can reprogram embryonic rat tracheal epithelium to express a distal lung phenotype. We have also demonstrated that embryonic rat lung epithelium can be induced to proliferate and differentiate in the absence of lung mesenchyme. In the present study we used a complex growth medium to induce proliferation and distal lung epithelial differentiation in embryonic tracheal epithelium. Day-13 embryonic rat tracheal epithelium was separated from its mesenchyme, enrobed in growth factor-reduced Matrigel, and cultured for up to 7 days in medium containing charcoal-stripped serum, insulin, epidermal growth factor, hepatocyte growth factor, cholera toxin, fibroblast growth factor 1 (FGF1), and keratinocyte growth factor (FGF7). The tracheal epithelial cells proliferated extensively in this medium, forming lobulated structures within the extracellular matrix. Many of the cells differentiated to express a type II epithelial cell phenotype, as evidenced by expression of SP-C and osmiophilic lamellar bodies. Deletion studies showed that serum, insulin, cholera toxin, and FGF7 were necessary for maximum growth. While no single deletion abrogated expression of SP-C, deleting both FGF7 and FGF1 inhibited growth and prevented SP-C expression. FGF7 or FGF1 as single additions to the medium, however, were unable to induce SP-C expression, which required the additional presence of serum or cholera toxin. FGF10, which binds the same receptor as FGF7, did not support transdifferentiation when used in place of FGF7. These data indicate that FGF7 is necessary, but not sufficient by itself, to induce the distal rat lung epithelial phenotype, and that FGF7 and FGF10 play distinct roles in lung development.

A 50-kDa variant form of human surfactant protein D.

The dominant form of human surfactant protein D (SP-D) is a multimeric collagenous glycoprotein composed of monomeric subunits that have a molecular mass of 43 kDa under reducing conditions. However, in evaluating monoclonal antibodies to human SP-D, an additional monomeric subunit was identified with a reduced molecular mass of 50 kDa. This 50-kDa variant was detected in approximately half of the samples evaluated and was found in lavage fluid from normal subjects, patients with alveolar proteinosis or idiopathic pulmonary fibrosis and in amniotic fluid. This 50-kDa variant had the same amino-terminal sequence, amino acid composition and apparent size of the carboxy-terminal collagenase-resistant fragment (20 kDa) as the 43-kDa subunit. The major difference was in the amino-terminal portion of the molecule and was due to altered glycosylation, as determined by carbohydrate staining, chemical deglycosylation, treatment with N-glycanase and neuraminidase and reduced signals for threonine at positions 5, 9 and 10 during amino-terminal sequencing. After gel filtration chromatography, the 50-kDa form was not present in the high molecular weight fraction, which is commonly used in purification of SP-D, but was found only in the smaller molecular weight fraction of monomers and trimers of SP-D. In conclusion, the 50 kDa-form of surfactant protein D is produced by post-translational glycosylation and does not form higher ordered oligomers, but its precise physiological function remains to be determined.

Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea.

Normal lung morphogenesis and cytodifferentiation require interactions between epithelium and mesenchyme. We have previously shown that distal lung mesenchyme (LgM) is capable of reprogramming tracheal epithelium (TrE) from day 13-14 rat fetuses to branch in a lung-like pattern and express a distal lung epithelial phenotype. In the present study, we have assessed the effects of tracheal mesenchyme (TrM) on branching and cytodifferentiation of distal lung epithelium (LgE). Tracheae and distal lung tips from day 13 rat fetuses were separated into purified epithelial and mesenchymal components, then recombined as homotypic (LgM + LgE or TrM + TrE) or heterotypic (LgM + TrE or TrM + LgE) recombinants and cultured for 5 days; unseparated lung tips and tracheae served as controls. Control lung tips, LgM + LgE, and LgM + TrE recombinants all branched in an identical pattern. Epithelial cells, including those from the induced TrE, contained abundant glycogen deposits and lamellar bodies, and expressed surfactant protein C (SP-C) mRNA. Trachea controls, and both TrM + TrE, and TrM + LgE recombinants did not branch, but instead formed cysts. The epithelium contained ciliated and mucous secretory cells; importantly, no cells containing lamellar bodies were observed, nor was SP-C mRNA detected. Mucin immunostaining showed copious production of mucous in both LgE and TrE when recombined with TrM. These results demonstrate that epithelial differentiation in the recombinants appears to be wholly dependent on the type of mesenchyme used, and that the entire respiratory epithelium has significant plasticity in eventual phenotype at this stage in development.

Influence of the cytoskeleton on surfactant protein gene expression in cultured rat alveolar type II cells.

We have investigated the role of the cytoskeleton in surfactant protein gene expression. Cytochalasin D (CD), colchicine (Col), or nocodazole (Noco) were tested on primary cultures of adult rat alveolar type II cells. Treatment with any of the drugs did not result in dramatic cell shape changes, but ultrastructural examination revealed that the cytoplasm of cells treated with CD was markedly disorganized; cells treated with Col did not exhibit such changes. Treatment with any of the three drugs resulted in a reduction in surfactant protein (SP) mRNAs. These decreases were not the result of cell toxicity, since overall protein synthesis was unimpaired by drug treatment. Washing the cells followed by an additional 2 days of culture resulted in a reaccumulation of SP mRNAs in CD-treated cells but not in Col-treated cells. Washing of Noco-treated cultures resulted in partial recovery. SP mRNA stability was estimated in the presence or absence of cytoskeleton-disrupting drugs. Disruption of either microfilaments or microtubules significantly affected the half-lives of mRNAs for SP-A, SP-B, and SP-C. These data support a role for the cytoskeleton in the maintenance of type II cell differentiation and suggest that the role of the cytoskeleton is at least in part to stabilize SP mRNAs.

Collagen matrices attenuate the collagen-synthetic response of cultured fibroblasts to TGF-beta.

Transforming growth factor-beta, a potent modulator of cell function, induces fibroblasts cultured on plastic to increase collagen synthesis. In 5- and 7-day porcine skin wounds, which have minimal to moderate collagen matrix, respectively, transforming growth factor-beta and type I procollagen were coordinately expressed throughout the granulation tissue. However, in 10-day collagen-rich granulation tissue type I procollagen expression diminished despite persistence of transforming growth factor-beta. To investigate whether collagen matrix attenuates the collagen-synthetic response of fibroblasts to transforming growth factor-beta, we cultured human dermal fibroblasts in conditions that simulate collagen-rich granulation tissue. Therefore, human dermal fibroblasts were suspended in attached collagen gels and collagen and noncollagen production was assayed in the absence and presence of transforming growth factor-beta. Although transforming growth factor-beta stimulated collagen synthesis by fibroblasts cultured in the collagen gels, these fibroblasts consistently produced less collagen than similarly treated fibroblasts cultured on plastic. This phenomenon was not secondary to nonspecific binding of transforming growth factor-beta to the collagen matrix. Fibroblasts cultured in a fibrin gel responded to transforming growth factor-beta similarly to fibroblasts cultured on plastic. Using immunofluorescence probes to type I procollagen, we observed that transforming growth factor-beta increased type I procollagen expression in most fibroblasts cultured on plastic, but only in occasional fibroblasts cultured in collagen gels. From these data we conclude that collagen matrices attenuate the collagen synthetic response of fibroblast to transforming growth factor-beta in vitro and possibly in vivo.

Modulation of alveolar type II cell differentiated function in vitro.

We have investigated whether the loss of differentiated function observed in adult rat alveolar type II cells cultured on a substratum that promotes cell spreading and flattening represents a reversible phenotypic change. Cells were cultured for 4 and 8 days in association with fetal rat lung fibroblast feeder layers on either attached collagen gels, which promote the loss of differentiated function, or on floating collagen gels, which support differentiation. A fifth group of cultures were maintained as attached gels for 4 days, then detached and cultured as floating gels for the remaining 4 days. Expression of mRNAs for surfactant proteins A, B, and C, patterns of phospholipid biosynthesis, rates and patterns of protein synthesis, and cell morphology were evaluated as markers of differentiation. Without exception, detaching the gels after 4 days in culture resulted in significant recovery of differentiated characteristics, demonstrating that type II cells modulate differentiated function in response to the culture environment. The results are discussed in relation to the importance of normal cell architecture to normal cell function and to the possible in vitro progression of type II cells to type I cells.

Transforming growth factor-beta stimulates the expression of fibronectin by human keratinocytes.

Transforming growth factor beta (TGF-beta) is a 25-kD protein which has regulatory activity over a variety of cell types. It is distinct from epidermal growth factor (EGF) and EGF analogs, and exerts its action via a distinct receptor. Its effect on proliferation or differentiation can be positive or negative depending on the cell type and the presence of other growth factors. It also modulates the expression of cellular products. TGF-beta causes fibroblasts to increase their production of the extracellular matrix components, fibronectin and collagen. Human keratinocytes (HK) are known to have TGF-beta receptors. We wished to study the effect of TGF-beta on the production of extracellular matrix proteins by human keratinocytes in culture. Human keratinocytes were grown in serum-free defined medium (MCDB-153) to about 70% confluence. Following a 16-h incubation in medium lacking EGF and TGF-beta, cells were incubated for 12 h in medium containing varying concentrations of EGF and TGF-beta. Cells were then labeled with 35S-methionine for 10 h in the same conditions. Labeled proteins from the medium were analyzed by SDS-PAGE and autoradiography. TGF-beta at 10 ng/ml induced a sixfold increase in the secretion of fibronectin, as well as an unidentified 50-kD protein. Thrombospondin production was also increased, but not over a generalized twofold increase in the production of all other proteins. EGF, at 10 ng/ml, caused a smaller additive effect. TGF-beta may be an important stimulator of extracellular matrix production by human keratinocytes.

Fibronectin extracellular matrix assembly by human epidermal cells implanted into athymic mice.

Human epidermal keratinocytes reorganize into epidermal inclusion cysts when implanted subcutaneously into athymic mice. During the organization and maturation of these cysts, fibronectin accumulates in the surrounding extracellular matrix and the basement membrane proteins bullous pemphigoid antigen and laminin appear at the epithelial-stromal interface. The sequence in which these proteins appear parallels that seen during reepithelialization of a skin wound in vivo. Fibronectin appears during aggregation of the epidermal cells and persists in the area surrounding the cysts for at least 7 days. Bullous pemphigoid antigen and laminin appear later (by 4 and 7 days, respectively) and ultimately become organized into a continuous band at the periphery of the cyst. This distribution of bullous pemphigoid antigen and laminin at the stromal-epithelial interface persists at least 5 weeks, suggesting that the implanted epidermal cells are capable of developing and maintaining a stable basement membrane zone. Fibronectin, which is abundant in the matrix adjacent to the epidermal cysts and in the surrounding stroma during cyst organization and maturation, diminishes to undetectable levels by 5 weeks. While much of the fibronectin derives from the host tissues, species-specific antibodies to human fibronectin reveal that at least a portion of this protein is synthesized and deposited by the implanted epidermal cells.

Basal cell carcinomas grown in nude mice produce and deposit fibronectin in the extracellular matrix.

Epidermal cells in vitro produce and deposit fibronectin (FN) in the pericellular matrix. Such FN production by epidermal cells may be involved in vivo in wound reepithelialization, tissue morphogenesis, and growth of epithelial tumors. The purpose of this study was to examine whether the FN, previously shown to be within and surrounding human basal cell carcinoma (BCC) lobules, was in part the product of epidermal-derived tumor cells. To examine this question we took advantage of our ability to grow human BCC in nude mice. Since we could demonstrate that all stromal cells surrounding the BCC were of mouse origin, antibodies specific for human FN would distinguish epithelial-derived FN from mesenchymal-derived FN. Five solid BCCs were implanted subcutaneously in nude mice. Growing tumors were removed after 60 days, snap-frozen, sectioned on a cryostat, and verified microscopically as BCC. The Hoescht DNA stain, which can distinguish mouse and human nuclei, demonstrated that mouse, not human, fibroblasts occupied the stroma surrounding each tumor lobule. Sections of all 5 BCCs were stained by immunofluorescence and immunoperoxidase techniques with antibodies to bullous pemphigoid (BP) antigen, laminin (LM), and FN. BP antigen and LM were present at the basement membrane zone (BMZ) of all tumor lobules as previously described for in situ BCC. FN staining was present along the BMZ, within the tumor lobules, and in the surrounding stroma. Antibodies to human FN were passed over a mouse FN affinity column to absorb antibodies which cross-reacted with mouse FN. The resultant antibody preparation, which was specific for human FN in this system, continued to demonstrate FN along the BMZ and within the tumors, but failed to stain FN in more distant stroma. Epidermal-derived cells, therefore, can synthesize and deposit FN in vivo in adjacent extracellular matrix. We speculate that this FN matrix may facilitate growth of BCC in this model.

Either exogenous or endogenous fibronectin can promote adherence of human endothelial cells.

Recently, we have presented evidence that proliferating blood vessels produce and deposit fibronectin in situ during the angiogenesis of wound repair. This report extends these observations by demonstrating that human endothelial cells from both large and small vessels depend on fibronectin for their adherence in vitro. Endothelial cells were grown from human umbilical veins (HUVEC) by the method of Gimbrone and from the microvasculature of human omentum by the method of Kern, Knedler and Eckel. Second-passage cells were plated into microtitre wells that had been coated with 100 micrograms ml-1 of fibronectin, types I and III collagen, type IV collagen or laminin. After a 3-h incubation, adherent cells were solubilized with Zap-Isoton and quantified on a Coulter Counter. Under normal culture conditions HUVEC showed no preference for fibronectin substrates while microvascular cells always demonstrated a striking preference for fibronectin substrates. However, when HUVEC were exposed to 2.5 or 25 micrograms ml-1 of cycloheximide for 4 h before and during the adherence assays, the adherence to fibronectin was 50-200% greater than to types I and III collagen. Immunofluorescence studies showed that while HUVEC expressed a large quantity of surface fibronectin, microvascular cells expressed very little. Metabolic labelling studies confirmed that HUVEC cultures had substantial quantities of fibronectin in their cell layer while microvascular cells did not. In antibody blocking experiments, preincubation of fibronectin-coated surfaces with anti-fibronectin antibodies totally blocked microvascular cell adhesion but only abrogated HUVEC adherence by 50%, presumably since these latter cells were able to deposit additional fibronectin onto the surface during the 3 h assay period. In the presence of cycloheximide anti-fibronectin antibodies totally blocked HUVEC adherence. These results demonstrate that both endothelial cell types rely, at least in part, on fibronectin for adherence in vitro. HUVEC can synthesize, secrete and deposit enough fibronectin for their adherence in vitro, while microvascular cells rely on an exogenous source of fibronectin under these culture conditions. Thus, the increased blood vessel fibronectin observed during angiogenesis in vivo may mediate adherence of the proliferating and migrating endothelial cells.

Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix.

Fibronectin and laminin production by human keratinocytes cultured in serum-free, low-calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7-day keratinocyte cultures was determined by 18 hr 35S-methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.

Immunological evaluation of a component isolated from Mycobacterium bovis BCG with a monoclonal antibody to M. bovis BCG.

A component of Mycobacterium bovis BCG referred to as BCG-a was isolated through the combined use of monoclonal antibody directed to BCG and affinity chromatography. Analysis of BCG-a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single prominent band with a molecular weight of ca. 10,000. Structural characterization of BCG-a consisting of amino acid composition and amino-terminal sequence determination was carried out. The intact BCG-a antigen was bound by neither the lectin from common lentils nor concanavalin A, implying that BCG-a does not carry any asparagine-linked oligosaccharides. Immunoprecipitation of 125I-labeled BCG-a with polyclonal and monoclonal antibodies directed against BCG resulted in bands having the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as did free 125I-BCG-a. In radioimmunoassays 125I-BCG-a was bound by the monoclonal antibody and by polyclonal antibodies from rabbits that had been immunized to BCG and to Mycobacterium tuberculosis H37Rv. Antibodies to nontuberculous and to nonacid-fast bacteria bound BCG-a poorly or not at all. The binding of 125I-BCG-a by the monoclonal antibody was readily inhibited by extracts of BCG and H37Rv, but it was not as readily inhibited by extracts of nontuberculous mycobacteria and was not at all inhibited by extracts of nonacid-fast bacteria. Considerable inhibition was similarly observed by surface antigens of nonviable, intact BCG organisms. Delayed cutaneous hypersensitivity reactions to small concentrations of BCG-a were elicited in guinea pigs that had been immunized with BCG or H37Rv antigens, but such reactions were not elicited in unimmunized animals.

Enhanced immunogenicity of line 10 guinea pig hepatocarcinoma cells after culture.

Line 10 hepatocarcinoma cells derived from ascites in a strain 2 guinea pig were tumorigenic when transferred intradermally. After they had been cultured in vitro for 20 days or more in medium enriched with 10% fetal bovine serum (FBS), they became immunogenic. Injections of immunogenic cells did not cause lethal tumors, and recipients were resistant to subsequent challenges with tumorigenic line 10 cells. Resistance was specific since growth of line 1 cells, a syngeneic but antigenically distinct tumor, was not affected. Cells cultured in medium enriched with 10% calf bovine serum or 10% normal guinea pig serum or in reduced concentrations of FBS were less effective in inducing resistance. When cultured line 10 cells were injected ip into normal guinea pigs, ascites tumors developed that were tumorigenic. The growth rate of line 10 cells in culture was considerably decreased as determined by reduced [3H]thymidine incorporation and mitotic indices. The mechanism(s) responsible for enhancement of immunogenicity in cultured line 10 cells is discussed but was not determined.