New brain stem and bone marrow abnormalities in victims of sudden infant death syndrome.

The study looked for new abnormalities in 31 victims of sudden infant death syndrome (SIDS). The focus was on respiratory control centers in the brain stem, because some SIDS victims have had abnormalities in respiratory control during sleep. A major respiratory control area (lateral reticular nucleus) of the medulla was hypomyelinated in 9 of the 31 SIDS victims. In a second study, the size of the 12th cranial nerve nucleus and its neuronal composition were analyzed because this nucleus regulates tongue movements, and the tongue has been postulated to help obstruct the airway in some SIDS victims. The 12th nucleus was found to have a neuronal deficit in more than two thirds of the SIDS victims. Finally, the SIDS victims were found to have a normoblastic hyperplasia in their bone marrows, a presumed response to chronic hypoxemia during sleep.

Immunocytochemical evaluation of primary human esophageal carcinomas and their xenografts for keratin, beta-chorionic gonadotropin, placental lactogen, alpha-fetoprotein, carcinoembryonic antigen, and nonspecific cross-reacting antigen.

The unlabeled antibody peroxidase-antiperoxidase technique was used to examine esophageal neoplasms for the tumor markers beta-human chorionic gonadotropin, human placental lactogen (HPL), alpha-fetoprotein, carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) before and after xenotransplantation to athymic nude mice. In addition, keratin was used as an epithelial cell marker. Immunoreactive beta-human chorionic gonadotropin was detected in four of seven primary tumors and in three of seven xenografts. Two of seven primary tumors contained HPL immunoreactive cells while four of seven tumor xenografts had HPL immunoreactivity. alpha-Fetoprotein was detected in two of seven primary tumors and in one of seven xenografts. NCA and CEA were detected in six of seven primary tumors and in all tumor xenografts. Five of seven primary neoplasms and six of seven tumor xenografts were found to contain both NCA and CEA, while one tumor and its xenografts displayed only NCA immunoreactivity. All seven primary carcinomas displayed keratin immunoreactivity, but only six of the seven xenograft tumors showed keratin positive cells. When a tumor marker was detected in a primary tumor, it was usually found in at least some of the xenografts arising from that tumor. However, marker loss did occur with repeated passage of tumors in some cases. On the other hand, markers were expressed in xenografts which were not present in the corresponding primary tumor. In three instances, HPL was detected in xenografts derived from HPL negative primary carcinomas. This was also true for CEA and NCA in one case. These results show that tumor markers are expressed to varying degrees by tumors growing as xenografts in nude mice. In primary tumors, HPL is associated with poorly differentiated squamous cell carcinomas and this marker was found to appear in HPL negative tumors as the tumor cells became less differentiated while growing as xenografts in nude mice.

Immunocytochemical evaluation of human esophageal neoplasms and preneoplastic lesions for beta-chorionic gonadotropin, placental lactogen, alpha-fetoprotein, carcinoembryonic antigen, and nonspecific cross-reacting antigen.

Human esophageal neoplasms were studied in comparison to normal, uninvolved, and preneoplastic human esophageal epithelium for the presence of human chorionic gonadotropin (HCG), human placental lactogen (HPL), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) using the unlabeled antibody peroxidase-antiperoxidase technique. HCG immunoreactivity was identified in 10 of 33 squamous cell carcinomas (33%), in 1 of 6 adenocarcinomas (17%), and 1 of 6 preneoplastic esophageal lesions (17%); while 9 of 33 squamous cell carcinomas (33%) and 1 of 6 adenocarcinomas (17%) contained immunoreactive AFP. Immunoreactive HPL was detected in 6 of 33 squamous cell carcinomas (20%), but in none of the adenocarcinomas. Neither AFP nor HPL immunoreactivity was identified in the 6 hyperplastic lesions which were studied. When stained with an antiserum that was able to detect both CEA and NCA, 27 of 33 squamous cell tumors (82%) and 6 of 6 adenocarcinomas (100%) showed positive immunostaining reactions. Of these, 8 squamous cell carcinomas and 1 adenocarcinoma were subsequently shown to contain only NCA immunoreactivity, while 19 squamous cell carcinomas and 5 adenocarcinomas contained both NCA and CEA immunoreactivity. NCA immunoreactivity alone was identified in 3 of 6 preneoplastic lesions and NCA and CEA immunoreactivity in 1 of 6 preneoplastic lesions. None of the markers was detected in 8 specimens of normal esophageal epithelium which were studied as controls, nor in 6 specimens of uninvolved esophageal epithelium obtained from patients with esophageal cancer. Most tumors expressed 2 or 3 markers, and some tumors were identified which expressed up to 4 of the 5 markers investigated. Only 3 tumors failed to express any of the markers studied. No association was found between the degree of tumor differentiation and presence or absence of HCG immunoreactivity. However, HPL immunoreactivity was more common in poorly differentiated squamous cell carcinomas. In contrast, immunoreactive AFP was more common in well-differentiated squamous cell carcinomas than in other tumor types. Similarly, both CEA and NCA were more frequently expressed in well-differentiated squamous cell carcinomas, adenosquamous carcinomas, and adenocarcinomas than in less differentiated tumors. Our results suggest that HCG, HPL, AFP, CEA, and NCA are tumor-associated antigens in esophageal cancer. Therefore, they could be of value in screening tests for esophageal neoplasms and could be useful in subclassification of esophageal neoplasms.

A computerized microspectrophotometry using fibre optics for transmission and detection of light.

A computerized microspectrophotometer was developed to provide rapid and accurate sequential measurements on cells, including nuclear DNA content, by fluorescence staining, photometric grain counting from autoradiographs, and nuclear size estimated by measurement aperture area. To alleviate bulk and vibrations caused by additional stepper motors, the excitation light and filters as well as the emission filters and photomultipliers were detached from the main microscope frame. The light was directed to and from the microscope through fibre optic bundles. This configuration provided an excellent light gathering faculty and made optical alignment easier. The machine provided highly accurate sequential measurements on a relatively large numbers of cells by static photometry.

The cell kinetics of the adaptation of the human esophagus to organ culture.

The adaptation of normal human esophageal explants to organ culture for the first 33 d of in vitro growth was evaluated using histomorphology and [3H]TdR autoradiography combined with mitotic blockade. On the 3rd d in culture, extensive desquamation of superficial cells reduced the epithelium to about four cell layers. Thereafter, the epithelium remained atrophic, with a relative increase in basal and suprabasal cells. The percentage of cells synthesizing DNA was greatest from Day 4 through 8, just after desquamation, and reached a maximum on Day 4 (24 h [3H]TdR labeling index of 62%). The labeling index (LI) fluctuated, thereafter, but remained high (26% on Day 33). During the last 6 h of each [3H]TdR labeling interval, mitosis was blocked by colcemid. The 6 h mitotic rate (MR) was a reasonably constant fraction of the LI (maximum at 4 d: MR = 1.44%), but was much lower than predicted by [3H]TdR labeling indicating the loss of large numbers of cells after DNA synthesis but before or during mitosis. Unlabeled mitotic figures appeared between Days 1 to 3 and 6 to 33, suggesting that the epithelium initially contained a considerable population of cells arrested or delayed in G2 and continued to generate cells that remained in premitosis longer than 24 h. These results indicate that the atrophy observed in vitro is characterized by a relative increase in the basal and suprabasal cell category, a high replication rate, initial recruitment of cells arrested in premitosis, and rapid cell turnover with significant loss of cells at the premitotic or mitotic step, or both. Thus it seems that human esophageal epithelium grown in organ culture is a satisfactory substrate for experimentation (for example, in vitro carcinogenesis) that requires cell replication. However, there are major differences between the kinetics of esophageal epithelium in vivo and in vitro.

Hemolytic-uremic syndrome: initial presentation mimicking intestinal intussusception.

A case of hemolytic-uremic syndrome preceded by laparotomy for suspected intestinal intussusception is reported. The pathologic lesions in the colon are described; they are attributable to microangiopathic thrombosis. Their significance in the hemolytic uremic syndrome is discussed.

A quantitative light and electron microscopic study of hamster tracheal epithelium with special attention to so-called intermediate cells.

Epithelial cells of the lower trachea of the hamster were studied and quantified by high resolution light microscopy and electron microscopy. The epithelium was composed of basal cells, secretory cells, and ciliated cells; preciliated cells were extremely rare in the undisturbed epithelium. In addition, a few cells were of indeterminate character. Correlative light and electron microscopy indicated that about 10% of the basal cell population, categorized by light microscopy, was composed of the nucleated basal portions of tortuous secretory cells. Electron microscopy revealed that the light microscopic indeterminate category was heterogeneous, being composed of several cell types, including secretory cells with scant secretion granules, some "tall" basal cells, and very few cells that were truly indeterminate. Furthermore, review of the literature and the results of this study indicated that several different cell types, including secretory cells with sparse secretions and preciliated cells, have been previously called "intermediate cells." The study focuses attention upon the difficulties in accurately classifying certain types of tracheal epithelial cells, especially at the light microscopic level.

Changes in secretory cells of hamster tracheal epithelium in response to acute sublethal injury: a quantitative study.

Secretory cells are reported to rapidly increase in number when the tracheobronchial epithelium is irritated. We suggest that this acute change results from accumulation of secretion granules within specialized albeit unobstrusive secretory cells rather than from conversion of undifferentiated cells to secretory cells of the production of new cells by mitosis. This hypothesis was tested in hamster tracheal epithelium 6, 12, and 24 hr after intratracheal instillation of elastase in saline or saline alone. Basal, secretory, and ciliated cells and cells of indeterminate character were quantified by counting 1400 cells per hamster in 2 micrometers thick glycol methacrylate sections stained with periodic acid-Schiff (PAS)-lead hematoxylin. Secretory cells were characterized and quantified depending upon distribution patterns of the PAS-positive secretion granules and on the amount of intracellular secretion product. Thirty-two hamsters were studied; half of them received colchicine 6 hr prior to tissue sampling. Over 24 hr the percentage of ciliated cells showed no significant change and no morphological evidence of alteration was observed in this cell population; thus ciliated cells were excluded from the analysis. When the ciliated cell population was excluded, control and experimental values for the different cell categories remained statistically constant, if unavoidable classification errors at the light microscopic level were compensated for on the basis of ultrastructural features. Average control values for the non-ciliated cell population were: mitotic index 0.030, basal cells 27.8%, secretory cells 57.9%, and cells of indeterminate character 14.3%. Secretory product was decreased in secretory cells at 6 hr but thereafter secretion accumulated in these cells. At 24 hr the overall number of secretory cells appeared to be increased. However, the increase was apparent and not real and was accounted for by accumulation of secretion granules in preexisting but previously unobtrusive secretory cells.

Probability paper analysis of flow cytofluorometric measurements of cellular deoxyribonucleic acid content.

A graphic analysis of cellular deoxyribonucleic acid (DNA) histograms obtained by flow cytofluorometry is described. This technique utilizes probability paper that graphs data in cumulative percentile form. From this graphic representation, individual normal distributions contributing to the histogram may be isolated and statistically describe. G0G1 and G2M distributions are independently described and S phase is fit to 1--5 overlapping normal distributions as dictated by the analysis results. This technique offers several advantages, including relatively simple mathematical calculations and few assumptions or restraints placed on the analysis. It has proved to be useful in analyzing proliferating and nonproliferating populations as well as distributions with unusual findings such as debris or aberrant DNA contents.

Variation in the autoradiographic technique: I. Emulsion-developer combinations assessed by photometric measurement of single silver grains.

Five commonly used developers (Acufine, Rodinal, Dektol, D19 and Microdol X) and two popular nuclear gel emulsions (Kodak NTB-2 and Ilford L4) were tested in combinations for adequacy of development characteristics and the distribution of silver particle (grain) size measured by means of a reflectance microphotometer. All developer-emulsion combinations had some range of development time during which maximum development was accomplished with no occurrence of background grains, with the exceptions of the combinations of NTB2-Acufine and NTB2-D19. Ilford L4 emulsion obtained the narrowest grain size distribution with Rodinal and Acufine, followed by Dektol, and D19. The narrowest size distribution for Kodak NTB2 emulsion was achieved with D19 followed by Acufine, Dektol and Rodinal. Microdol-X had undesirable effects on the integrity of the individual grains of both emulsions. The criteria are discussed for selecting the most advantageous emulsion-developer system for the particular mode of evaluation (visual, photometric, television camera) to be applied to the finished autoradiographic specimens.

Tumors of the pilosebaceous unit induced in the rat by the intravenous administration of 7,12-dimethylbenz(A)anthracene.

Six of 70 female Sprague-Dawley rats given a single intravenous injection of 7,12-dimethylbenz[a]anthracene (DMBA) developed 7 pilosebaceous tumors. Two of the tumors showed differentiation toward the upper portion of the pilosebaceous unit while 5 showed differentiation toward the lower portion. Each tumor was examined histochemically for the presence of inner root sheath keratin of the hair follicle using the carbamido diacetyl reaction for citrulline and for hair shaft keratin using boiling ninhydrin reagent. The 2 tumors of the upper portion of the pilosebaceous unit were sebaceous adenomas which were accompained by a keratinizing epithelim like that of the sebaceous gland duct and upper pilosebaceous canal. Histochemically, the keratin was not like that of hair shaft nor inner root sheath. The 5 tumors showing differentiation toward components of the lower pilosebaceous unit were trichoepitheliomas. They were composed of structures which, to varying degrees, recapitulated the organization of the normal hair follicle. Within these follicular structures, both inner root sheath and hair shaft type keratins were found. The occurrence of skin tumors after the intravenous administration of DMBA was unexpected since it is uncommon for skin tumors to be produced by the systematic administration of chemical carcinogens and they have never been described after the oral administration of DMBA. That the route of administration may influence tumor production with this carcinogen is suggested by the fact that the only other reported tumors, which were squamous carcinomas, also followed intravenous injection of DMBA.