[1] 25w The irregular, skeinlike appearance of the hyaline bodies clearly differentiated them from giant mitochondria, which are occasionally visible by light microscopy as regular, circumscribed, round,

[1] 158w Abstract. Serums from tumor-bearing patients, cured patients, and normal sub- jects were examined for antibodies to the separated complement-fixing reactive components of nonvirion antigens of herpesvirus type I and type 2. The occur- rence of antibodies to the antigens was similar in serums from tumor-bearing patients and cured patients. Antibodies to the antigens were observed among 21 of 24 (87 percent) cervical cancer cases, 44 of 49 (90 percent) laryngeal cancer cases, 15 of 24 (62 percent) cases of squamous cell carcinomas of the head and neck excluding the larynx, 2 of 24 (8 percent) nonsquamous cell cancer cases, and 3 of 51 (6 percent) normal subjects. By contrast, no differences were found in the titers of neutralizing antibodies to the virus in serums from laryngeal cancer patients and controls. The observations support an etiologic role of herpesviruses in cervical cancer and in laryngeal cancer, and possibly other squamous cell * Includes some subjects shown in Table 1.

[2] 25w t Specific cancer sites with number positive/number tested: tongue, 3/8; mouth and palate, 4/5; tonsil, 4/4; maxillary sinus, 3/3; pyriform sinus, 1/3; and gingiva, 0/1.

[3] 29w t Nonsquamous cell cancers include cases of melanoma (10), breast (4), salivary gland (4), rectum and colon (3), lacrimal gland (1), pancreas (1), and chondrosarcoma of the mandible (1).

[4] 94w § Cured are patients who are disease-free four or more years after treatment. is designated region 3, reacts specifically with absorbed antiserums to HSV nonvirion antigens (6, 7). Although the soluble portions of the tumor antigens and the nonvirion antigens are different in composition, there are shared components of identity necessary for CF reactivity present in the gel regions 3 of both the preparations. Since the amount of antigen present in tumors varies, we chose to separate gel regions 3 from HSV type 1 (KOS strain) and HSV type 2 (SAV strain) nonvirion antigens.

[5] 109w Cultured human fetal kidney cells infected with HSV (35 plaque-forming units per cell) were harvested at 24 hours, disrupted with sonication (1 minute), and centrifuged at 100,000g for 1 hour. The pellets of both infected and noninfected cells were sequentially sonicated (low frequency, at 3, 1.5, and 1.5 minutes) and centrifuged at 100,000g. The supernatants were pooled, concentrated by ultrafiltration, and separated by PAGE (3, 6, 7). Separation by Sephadex gel ifitration prior to PAGE as described earlier (1, 6) could be omitted. Region 3 of each gel was sliced, the protein was eluted, and the eluates were concen- trated to uniform protein content by ultrafiltration and tested immediately.

[6] 202w Serums were collected in Houston, Texas, between October 1971 and June 1972 and stored at -35°C; the de- mographic attributes of these patients are described elsewhere (8). The Na- tional Cancer Institute (NCI) serums were collected within the past 3 months, passed through Millipore filters (0.4 ,um), and stored in liquid nitrogen. Donors of serums collected at NCI were screened to exclude those receiv- ing chemotherapy or radiotherapy or those with illnesses or on medications known to alter immune reactivity. The titers of antigens, complement, and serums were ascer,tained before use at selected concentrations in a standard macro CF test (1). Complement dilu- tions varied from 1 :15 to 1 :20 and 714 serums were tested at a dilution of 1:4. The region 3 protein content was regu- lated to 50 jug per CF test for the serums from Baylor University; some positive CF tests were seen at this same protein concentration with the filtered serums from NCI, but it was necessary to repeat this test with 80 jtg of protein per CF test; the ratio of CF positive cancer serums to CF positive control serums was similar for both tests. All tests were uncoded after the completion of the final experiments.

[7] 120w Initially, serums from patients with laryngeal cancer and control subjects (8) were examined for antibodies to HSV nonvirion (from PAGE region 3) antigens, to region 3 cell antigens, and to virion antigens (Table 1). Anti- bodies to nonvirion antigens were found in 36 of 38 (95 percent) patients with laryngeal cancer and 2 of 36 (5 percent) control patients. No reactions were seen with control PAGE region 3 prepara- tions. There were no differences be- tween cancer cases and controls in antibodies to virus, as assayed by CF or by a microneutralizaton technique (9). Thus, the presence of antibodies to the HSV nonvirion antigens is associated with the laryngeal cancer and does not simply represent past infections with the herpesviruses.

[8] 149w To further clarify the relations of antibodies to the nonvirion antigens, we examined another group of patients with different types of malignancies at various sites. Some of this group were tumor-bearing patients, and others had been cured of their disease. Among squamous cell cancer patients, little difference was found between tumor- bearing patients and those clinically cured of cancer (Table 2); serums from 48 of 57 (84 percent) tumor- bearing and 20 of 28 (71 percent) cured patients showed CF reactivity with nonvirion -antigens of region 3. Antibodies to the nonvirion antigens were present in the serums of 21 of 24 (87 percent) women with carcinoma of the cervix. Only 2 of 24 (8 percent) patients with nonsquamous cell cancers had antibodies to the antigens. Serums from 15 of 24 (62 percent) patients with squamous cell cancers of the head and neck other than larynx reacted to the antigens.

[1] 46w or ovoid cytoplasmic globules (1). Ne- crotic zones, containing numerous ,mononuclear and occasional polymor- phonuclear inflammatory cells, were fre- quent around central veins. These areas showed conspicuous fibrosis, which oc- casionally obliterated the efferent veins. In some areas, thin fibrous septa con- nected adjacent central zones.

[2] 186w The entire constellation of histologic features characteristic of human alco- holic hepatitis has been produced for the first time in an experimental model. Moreover, the activity of serum trans- aminase was also increased, a common finding in alcoholic hepatitis. On the basis of early experiments with rats fed ethanol in drinking water (7), it was claimed by some that liver disease re- lated to alcohol abuse is caused by associated malnutrition, and that a nutri- tionally fortified diet protects against the deleterious effects of chronic alco- holism. The data presented here indi- cate that not only fatty liver, but also alcoholic hepatitis, is a direct result of alcohol toxicity, and is not related to nutritional factors, since the diets of all animals contained excess protein, min- erals, and vitamins. In man, alcohol- induced cirrhosis of the liver, often pre- ceded by alcoholic hepatitis, usually follows 10 to 15 years of excess alcohol consumption (8). It is, therefore, not surprising that this irreversible phase of liver disease was not seen after only 9 months. It will be of interest to see whether the baboons eventually develop cirrhosis.

[3] 59w 6. The animals consumed daily 78 to 94 mg/kg of a diet which contained the following in- gredients per liter: dl-a-tocopherol acetate, 30 mg; thiamine hydrochloride, 1 mg; pyridoxine hydrochloride, 1 mg; folic acid, 1 mg; calcium pantothenate, 3 mg; nicotinic acid, 5 mg; as- corbic acid, 25 mg; choline chloride, 100 mg; cancers of the head and neck.

[4] 182w Concentrates of purified antigens from viable cells from squamous cell carcinomas of the genital tract reacted in complement-fixation tests with anti- bodies present in the serums of patients with squamous cell cancers of the same site (1). With DNA tumor viruses, it has been possible to separate virus-induced, nonvirion antigens not only from tumors but also from cells under- going lytic infection by the viruses (2). Separated antigens from squamous cell carcinomas of both the lip and cervix react specifically with antibodies to herpesvirus (HSV) nonvirion antigens (3). Antibodies to HSV nonvirion anti- gens have been detected in serums from patients with cervical cancer (3,4), as well as patients with cancers of the lip (3), prostate, urinary bladder, kidney, and nasopharynx (5). Our study was undertaken to find out whether serums from patients with squamous cell can- cers at various sites and with non- squamous cell cancers contained anti- inositol, 100 mg; p-aminobenzoic acid, 100 mg; vitamin B12, 1 itg; vitamin A, 4000 international units (I.U.); vitamin D3, 400 I.U. 7. C. H. Best, W. S. Hartroft, C. C. Lucas, J. H.

[5] 71w Ridout, Br. Med. J. 2, 1001 (1949). 8. W. K. Lelbach, Acta Hepato-Splenol. 14, 9 (1967). 9. Supported in part by PHS grants AA00224, AA00287, and AM02511; the Veterans Administration; and the Laboratory for Experi- mental Medicine and Surgery in Primates. 28 June 1973; revised 30 July 1973 bodies to HSV nonvirion antigens. In addition, the influence of successful therapy on the presence of antibodies to the above antigens was evaluated.

[6] 160w The tumor antigens were separated by a special method of polyacrylamide gel electrophoresis (PAGE) into three gel regions; cancer gel region 3, near the anodal end of the gels, was positive for complement-fixing (CF) reactivity with the antiserums specific for HSV nonvirion antigens (6). With recent techniques of sequential, stepwise, low-frequency sonication, and subse- quent centrifugation, the soluble por- tion of HSV nonvirion antigens has been studied with several strains of the virus (7). Analysis by PAGE of each sonicated fraction reveals the slow ap- pearance of the genetic marker; after the final sonication all of the CF re- activity is present in the supernatant, with the final pellet being negative for CF reactivity (7). The portion of the supernatant separated by PAGE, which * Mean titers of neutralizing antibodies are as log1.. One case and one control had no neutralizing antibodies to either virus. t Reactions in complement-fixation test to type 1 or type 2 (or both) nonvirion antigen.

[7] 33w t 91 percent nonvirion antigen-positive serums from tumor-bearing and 100 per- cent nonvirion antigen-positive serums from patients up to 4 years after treatment. Primary sites were 21 percent supraglottic and 79 percent glottic.

[8] 8w Antibodies to Herpesvirus Nonvirion Antigens in Squamous Carcinomas

[9] 62w We observed no pattern of preferen- tial reactivity to the nonvirion antigens induced by type 1 HSV (HSV1) or type 2 HSV (HSV2). Serums reacted to only HSV1 antigen in 18 percent of the serums that had antibodies and to only HSV2 antigen in 5 percent of the serums which had antibodies. The re- maining positive serums reacted with both antigen preparations.

[10] 60w The high occurrence of antibodies to HSV nonvirion antigens among pa- tients with squamous cell carcinomas but not among normal subjects or pa- tients with nonsquamous cell cancers suggests an association between the HSV and squamous cell lesions. The finding of antibodies to the nonvirion antigens in serums of patients with cervical cancer confinrns previous re- ports (1, 3, 4).

[11] 129w Patients with laryngeal carcinomas were also found to have antibodies to the nonvirion antigens, suggesting an etiologic role of HSV in carcinomas of this site. Herpesvirus type 1 is primarily transmitted by the oral-respiratory route, while HSV2 is venereally transmitted. Laryngeal cancer cases were similar to controls in the sexual and reproduction-associated factors as- sociated with sexual promiscuity (8). This observation and the pattern of neutralizing antibodies to the two viruses shown in Table I suggests that the orally transmitted type 1 virus would be the most likely candidate. Antibodies to HSV nonvirion anti- gens were found among patients with squamous cell cancers of the head and neck other than the larynx, but the numbers studied were not sufficient to establish an association between the herpesviruses and each site.

[12] 35w Although most of the serums with antibodies to the nonvirion antigens re- SCIENCE, VOL. 182 acted to antigens induced by both HSV1 and HSV2, some serums reacted to one but not to the other antigen.

[13] 78w The antigens of the two types ap- pear to contain common and specific antigenic sites (7). The exact nature of the antigens and the relatedness be- tween the two types of HSV remain unknown. Certain cytomegalic virus strains have been isolated from primary lung cancer cell cultures; there are indi- cations that other members of the HSV group infecting humans induce similar antigens (10). The relation between the antigens of the HSV group, however, has not been established.

[14] 333w The continued presence of antibodies to the nonvirion antigens after success- ful treatment of the malignancy paral- leled the finding of a high incidence of cellular immune abnormalities in both tumor-bearing and cured squamous carcinoma patients (11). If the antibodies were produced to antigens re- lated specifically to the tumor cells, removal of the tumor should eliminate the antigen source and result in a sub- sequent disappearance of antibodies. However, removal of the malignancy may not eliminate all cells producing the antigens; clinically normal adjacent mucosa may, nevertheless, contain transformed cells. This may explain the well-documented, repeated development of new primary squamous cell carcinomas in these patients. On the other hand, cells in the premalignant stage of the disease might remain, or nonmalignant cells lytically infected with virus during recurrent infection might pro- vide an antigen source for continued antibody stimulation. For these reasons it is important to determine the rela- tions of antibodies to the antigens, to each other, and to the cancer. Similarly, a possible relation between expression of nonvirion antigens and the prolonged immune cellular abnormalities in the squamous cell cancer patients deserves attention. Further knowledge of the rates of antibody appearance and can- cer development is necessary before the techniques can be applied clinically. A common feature associated with patch reefs in the West Indies is a band of nearly bare sand between the base of the reef and the outlying beds of marine monocotyledons Thalassia testudinum (turtle grass) and Syrin- godium filiforme (manatee grass). This so-called halo is commonly 2 to 10 m in width and is reminiscent of the bare zones reported between California shrub and grassland communities (1). Generally, the area closest to the reef is totally barren. The portion of the halo closest to the outlying beds of sea grasses usually has a moderate density of closely cropped plants and appears bare when viewed from afar or in aerial photographs (2). Some attention has been paid to the origin of these halos. Previously it was

[15] 97w 16 40 12 30 6 Bad nt 10 ! 22\ -Di:d.ma. held that certain physical factors such as large particle size and rapidly shift- ing sands made growing conditions un- favorable in halos. However, it was found that halos were present over a wide range of physical environments. Neither the slope of the bare area, nor exposure to high-energy wave surge had any bearing on the presence or absence of the halo. Furthermore, anal- ysis of the sediment revealed little dif- ference in particle size between areas on the halo and areas in the grass beds (2).

[16] 124w Randall (2) presents some evidence that grazing fish-primarily surgeon- fish (Acanthuridae) and parrot fish (Scaridae)-are responsible for the ab- sence of vegetation in the halos. Recent observations have cast doubt on the extent to which herbivorous fish are responsible for halo formation and maintenance. First, grazing fish in the area of patch reefs on St. Croix, U.S. Virgin Islands, are under intense fish- ing pressure and are not common. Second, close observation of the short cropped grass within the halos sur- rounding these patch reefs shows that many of the blades do not display the scalloped edges characteristic of fish grazing. Rather, the blades are termi- nated in the ripped and jagged form which characterizes grazing by the echinoid Diadema antillarum Philippi (3, 4).

[17] 21w In the West Indies, D. antillarum is found in abundance on patch and bar- rier reefs (5). It remains inactive on