Hernan F. Acevedo

Immunohistochemical localization of a choriogonadotropin-like protein in bacteria isolated from cancer patients.

By the use of specific antibody to human chorionic gonadotropin (CG) as well as to its β‐subunit, and the application of the indirect fluorescein‐labeled and peroxidase‐labeled antibody techniques, we have demonstrated the presence of a membrane (wall)‐associated CG‐similar immunoreactive protein in 15 strains of bacteria isolated from tissues of patients bearing malignant neoplasms. These microorganisms were classified as S. epidermidis, (12), E. coli (2), and a single strain of P. maltophilia (ATCC 13637). The absence of the CG‐like antigen in other “cancer associated bacteria”, Streptococcus faecalis (ATCC 12818) and Pseudomonas aeruginosa (from patient with cancer of colon), demonstrated that not every “cancer associated bacteria” has the capability to synthesize the trophoblastic‐like protein. The negative results obtained with a number of “noncancer control” bacteria of known origin, obtained from ATCC and from clinical samples, strongly supported the idea that the existence of these CG‐like protein producing microorganisms is not a ubiquitous finding. The demonstration of a de novo bacterial biosynthesis of a protein having similar antigenic and biophysical properties to those of the human trophoblastic hormone, has great biological implications, especially if its biosynthesis is proven only in bacterial strains growing in the presence of cancer cells in which we have already demonstrated the presence of a similar antigen. The explanation of the phenomenon is unknown. Because of their origin, the potential of “genetic exchange” with subsequent expression of the mammalian gene by the bacterial cells becomes a possibility. It is also possible that the gene coding for the CG‐like protein is normally present but inactive or repressed in all bacteria.

Urinary cholesterol V. Its excretion in men with testicular and prostatic neoplasms

Urinary excretion of nonesterified cholesterol (NEC) in men with testicular and prostatic neoplasms has been investigated. Control patients with non‐steroid‐related benign and malignant tumors and patients with nonneoplastic diseases were also studied. Normal range of NEC excretion was determined in healthy individuals and was found to be 0.10‐1.20 mg/24 hours (3 S.D., 99.5% population), with a mean of 0.65 ± 0.18 mg/24 hours. Twenty‐nine of 32 patients with adenocarcinoma of the prostate (91%) had NEC hyperexcretion. In addition, all patients with choriocarcinoma, teratocarcinoma, and embryonal cell carcinoma of the tesds (8 cases) had NEC hyperexcretion. Hyperexcretion of NEC was also present in patients with metastatic carcinoma of testes and prostate in which the primary tumor had been removed. Testicular seminomas (19 cases) on the other, hand, revealed a normal urinary excretion of NEC. The effects of therapy on NEC excretion were investigated in some of these patients, and their NEC values appeared to correlate with the clinical course of the disease. Approximately 25% of the control patients over 45 years of age without histologically demonstrable testicular and/or prostatic carcinomas revealed NEC hyperexcretion. This hyperexcretion of NEC in the control groups can possibly be explained in part by the presence of “latent” adenocarcinoma of the prostate that was not detected. It is recognized that at least 30% of necropsies performed in men at this age group will harbor an unsuspected adenocarcinoma of the prostate. Our previous work in this field has shown NEC hyperexcretion in women with carcinomas of the steroid‐producing glands and their target organs. This suggests a certain cell specificity. The specificity of the results in both men and women, in respect to type of cells involved, effects of therapy, and lack of correlation of the NEC excretion with the serum levels of cholesterol, supports the concept that most of the urinary cholesterol is of endogenous origin, and appears to represent an expression of cell biochemical function.

Determination of urinary cholesterol by gas-liquid chromatography

Abstract A method suitable for the routine determination of cholesterol in human urine has been developed. The procedure consists of a simple chloroform extraction of a urine aliquot, acetylation, and subsequent gas-liquid chromatography (GLC) with an internal standard utilizing a peak-height ratio technique for quantitation. An assessment of the reliability of the method as well as excretion values in normal individuals are presented and discussed.

Urinary steroid profile as an index of fetal well-being

Abstract A study of the steroid metabolism of the fetoplacental unit in the human as reflected in the simultaneous analyses of total estrogens, pregnanetriol, pregnanediol, and pregnanolone in the same urine sample is presented. The study incorporates an automated assay for total estrogens and gas-liquid chromatography of the pregnane metabolites. The use of these highly sensitive and specific analytical tools allowed us to establish the pattern of excretion of the aforementioned compounds in a total of 170 normal and abnormal pregnancies in various stages of gestation. A total of 2,704 determinations were carried out in 14 months. One hundred and seven patients were considered normal, and the results from this group graphically presented gave the basis for “Fetal Health Survey” criteria of normalcy. Some of the results of the abnormal group, clinically classified, are discussed in terms of the normal pattern of excretion.

The determination of urinary estrogens in pregnancy using an automated fluorometric assay

Abstract A semi-automated method for the determination of total urinary estrogens in pregnant women has been developed. The procedure employs the salting-out of the urinary estrogens with ammonium sulfate as outlined by Cohen (1). The solutions made of the resulting precipitates are then assayed by an automatized Ittrich-Kober fluorescence reaction. This method meets the criteria of speed, accuracy and reproducibility needed for the assessment of fetal health and/or threatened abortion. In fetal health programs it can be applied to the mass screening of the pregnant status.

Effects of antibodies to choriogonadotropin in malignant growth: I. Rat 3230 AC mammary adenocarcinoma

To determine whether the in vivo production of antibodies against choriogonadotropin (CG) could modify the relationship between host and malignant growth, the effects of preimmunization with a conjugate of the β‐subunit of CG and tetanus toxoid (CGβ‐tt) on the development and growth of lung metastasis following I.V. injection of R 3230 rat mammary adenocarcinoma cells into Fischer 344 female rats have been investigated. This neoplasm has been previously demonstrated to synthesize CG‐like material. A test group of 70 animals and two matched control groups of 15 animals each were used. One of the control groups was untreated and the other received tetanus toxoid only. All the control animals had multiple lung foci of neoplastic cells ten days after seeding, as has been previously observed, and CG antibodies were undetectable. In contrast, a significant titer of anti‐CG was found in all preimmunized animals. At the same time, the pretreated animals rarely had a few small neoplastic nodes in the entire lung sections at the standard 8–10 days post‐seeding. The protective effects of preimmunization with CGβ‐tt were further demonstrated by the animals living 20 days post‐seeding, the absence of lung pathology in the ones killed thereafter, and by six animals that were left alive and have not shown any deterioration for more than six months after the administration of the cell suspension.

Urinary cholesterol. VIII. Its excretion in women with ovarian neoplasms.

The urinary excretion of nonesterified cholesterol (NEC) has been investigated in 57 women with ovarian neoplasms and/or related nonneoplastic diseases. Twelve patients had benign tumors or lesions and 45 had malignant neoplasms of their ovaries. All patients with nonmalignant ovarian tumors or lesions had normal NEC excretion irrespective of the type of tumor or lesion or its degree of extension. In contrast, urinary NEC hyperexcretion occurred with the following frequencies in patients with active malignant ovarian neoplasms: 18 of 19 cystadenocarcinomas of the serous and/or mucinous types; one of one endometrioid carcinoma; four of four malignant granulosa cell tumors; two of two mixed malignant germ cell tumors; and one of one malignant mixed müllerian tumor. Single cases of clear cell carcinoma and of rhabdomyosarcoma had a normal NEC excretion. Urinary hyperexcretion of NEC was also found after surgery in two of seven surviving patients with apparently localized resectable disease according to their staging. It is possible that in these two patients NEC hyperexcretion was due to undetected foci of cancer (wrong staging), since neither omental and peritoneal biopsies, nor cytologic examination of peritoneal washings or free fluid were performed. A normal excretion of urinary NEC has been characteristic of 19 of 21 surviving patients treated by surgery and adjunctive therapy in whom we have performed follow‐up NEC determinations. They were 16 of 18 cystadenocarcinomas, and single cases of endometrioid carcinoma, malignant granulosa cell tumor, and malignant germ cell tumor. The 94% correlation between the presence of proven active ovarian carcinomas and urinary NEC hyperexcretion is significant. The clinical significance of this investigation is even greater when one considers that cystadenocarcinomas constitute more than 75% of all primary malignant ovarian tumors.

Urinary cholesterol. VII. The significance of the excretion of nonesterified cholesterol in patients with uterine carcinomas

The urinary excretion of nonesterified cholesterol (NEC) in 170 women with cervical and endometrial carcinomas has been investigated. Control patients (236) included: 1) women with other types of benign and/or malignant diseases of the pelvic organs; 2) patients with non‐steroid‐related neoplasms; 3) patients with benign and/or malignant breast diseases other than carcinoma; and 4) patients with a variety of non‐neoplastic diseases. NEC was determined by a gas‐liquid chromatographic procedure. The range of NEC excretion for clinically healthy normal women (64) was previously established by this method. NEC hyperexcretion was defined as any NEC value over 1.5 mg/24 hours. The results showed NEC hyperexcretion in 65 of 68 women with active carcinoma of the cervix, including 13 patients with carcinoma in situ, and in 42 of 45 women with active carcinoma of the endometrium. In contrast, a normal excretion of NEC occurred in all the patients (77) of the first and second control groups, in 39 (80%) of the 48 patients of the third control group (high‐risk group), and in 101 of the 111 patients of the fourth control group. Sequential studies performed in patients with uterine carcinomas have demonstrated an almost perfect correlation between the NEC excretion and the clinical status of the patient following surgical and/or radiation therapy. Of 57 patients (31 cervix and 26 endometrium) in which the NEC studies were started after treatment was instituted, 53 have normal NEC excretion in the multiple determinations performed to date. Presently these patients have no clinical, chemical, or radiologic evidence of cancer. It is concluded that urinary NEC determinations can be used as an additional diagnostic biochemical test to detect active carcinoma of the steroid‐producing glands and their main target organs, and that in women with uterine carcinomas, the test can be used as an objective laboratory method to monitor the course of the disease and the response of the patient to therapy.

Urinary steroid profile in threatened abortion

Abstract A study of the urinary steroid profile in early pregnancy has been undertaken in order to re-evaluate the usefulness of the urinary determinations of pregnanolone, pregnanediol, pregnanetriol, and total estrogens in cases of threatened abortion. This investigation incorporates an enzymatic hydrolysis with gas-liquid chromatographic analysis for the pregnane metabolites and an automated assay for total estrogens. The results obtained in 62 patients confirm the value of the urinary determinations of pregnanediol and pregnanolone. They also suggest that the prognosis is good in cases which present clinical symptoms of threatened abortion but have normal urinary excretion of pregnanediol and pregnanolone. When the “biochemical progestational activity,” as evaluated by the analysis of pregnanolone and pregnanediol remains normal, abortion does not occur in the first trimester.

Urinary cholesterol—VI. Its excretion in women with inoperable inflammatory carcinoma of the breast

The urinary excretion of nonsterified cholesterol (NEC) in 14 women with inoperable inflammatory carcinoma of the breast has been investigated. Patients with breast diseases other than carcinoma as well as patients with a variety of nonsteroid‐related neoplasms comprised control groups. NEC was determined by the gas‐liquid chromatographic method of Vela and Acevedo.8 By this method, the range of NEC excretion for normal women was previously established as 0.35–1.50 mg/24 hr. Nine of 11 women with inoperable breast carcinoma demonstrated NEC hyperexcretion prior to radiation therapy. Four of 11 patients studied during the course of therapy showed a transient diminution of NEC to normal values of excretion. However, all patients evidenced NEC hyperexcretion after completion of therapy. Eleven of the original 14 patients have died. In the first control group, 7 of 42 patients with diseases of the breast other than carcinoma revealed NEC hyperexcretion before histopathologic diagnosis. Of these 7 patients, 2 had fibroadenoma, 4 had fibrocystic disease, and 1 had cystosarcoma phyllodes. In the second control group that comprised 46 women with nonsteroid‐related neoplasms, there was no NEC hyperexcretion regardless of age, type of cancer, or severity of the disease. The results parallel the finding of NEC hyperexcretion in previous studies of patients, both males and females, with histopathologically proven carcinomas of the steroid‐producing glands and their main target organs.