Antibody reacting with the murine mammary tumor virus in the serum of patients with breast carcinoma: a possible serological detection method for breast carcinoma.

Sera from patients with Stages A and B infiltrating ductal carcinoma of the breast, benign breast disease, cancers other than breast carcinoma, and normal female controls were examined by indirect immunoelectron microscopy (IEM) and a viral agglutination test for evidence of antibodies directed against murine mammary tumor virus (MMTV). Sera from 41 (79%) of 52 patients with breast carcinoma and eight (19%) of 42 normal subjects or patients with benign breast disease (noncancer subjects) showed evidence of MMTV labeling by IEM. In the MMTV agglutination test, significant virus agglutination (2+ to 4+) was present in eight (13%) of 61 noncancer sera, 58 (86%) of 68 breast carcinoma sera, and two (11%) of 18 other cancer sera. The results of the more rapid MMTV agglutination test correlated well with IEM. Analysis of reacting antibody by IEM revealed no immunoglobulin A and significant immunoglobulin M and immunoglobulin G antibody. Serum reactivity against MMTV was completely absorbed by MMTV but not by the glycoprotein with a molecular weight of 52,000 of MMTV, Friend murine leukemia virus, avian myeloblastosis virus, or sheep erythrocytes. It is concluded that reactivity of human antibodies to MMTV is strongly associated with, but is not entirely specific for, breast carcinoma. It remains to be determined if normal persons with these antibodies will ultimately develop breast cancer and should therefore be considered at high risk. These tests may have potential usefulness as a diagnostic screen for breast cancer.

Detection of the major glycoproteins of Friend leukemia virus (gp71) and the murine mammary tumor virus (gp52) on the surface of mouse cells.

Specific rabbit antisera to the major glycoproteins of Friend leukemia virus (gp71) and the mouse mammary tumor virus (gp52) were utilized to study the surfaces of C3H-, DBA-, BALB/c-, and C57BL-transformed and normal cells by immunoelectron microscopy. Antiserum to gp71 showed reactivity with all of the mouse cells tested regardless of strain, virus production, or state of transformation. In cells producing murine leukemia virus, budding viruses and other areas of the cell surface were consistently labeled with gp71 antiserum. A gp52-like antigen was likewise detected on both cell surfaces and virions of C3H and DBA cells producing the mammary tumor virus. Budding virions with surface spikes but with crescent-shaped nucleoids in C3H/HeJ cells were labeled specifically with gp52 antiserum. The antigen localized with anti-gp52 serum was detected in low concentration on the surface of nonvirus-producing cultures of a C57BL/6 sarcoma induced by the Schmidt-Ruppin D strain of Rous avian sarcoma virus (SRD-2), a BALB/c bone marrow culture (JLS-V9), and a normal BALB/c fibroblast culture (BALB/cF). Other cell cultures transformed by either C-type virus or methylcholanthrene failed to demonstrate gp52 antigen. Both gp52- and gp71-like antigens were found to be expressed simultaneously in C3H/HeJ, C3H-MT, DBA-MT, SRD-2 (transformed) and BALB/cF, JLS-V9, and C3H-1 (normal) cultures. Expression of gp52 antigen in the absence of gp71 was not detected in any of the cultures examined. These findings demonstrate the ubiquitous expression of gp71 in a wide variety of normal and transformed mouse cells while gp52 tends to be expressed predominantly in cells from mice with high mammary tumor incidence (C3H) and DBA), but only to a minor extent in cells from low mammary tumor incidence strains (BALB/c and C57BL).

Morphological and biochemical properties of a new human breast cancer cell line.

DU4475 is a new human breast cell line derived from a cutaneous metastatic nodule from a patient with advanced breast cancer. It has been in continuous culture for more than 1 year and has survived 140 subcultivations. The cells grow in suspension, displaying clusters and free-floating aggregates with serpentine-like outgrowths. The cell line is hypotetraploid with modal chromosome numbers between 86 and 93 and possesses five to six distinctive marker chromosomes. The cells grow readily in athymic nude mice and produce tumors from which cells can be reintroduced to culture in vitro.

Toxicity and immunologic effects of continuous infusion of recombinant human interleukin-2 administered by selective hepatic perfusion in dogs.

A preclinical pilot study was done to evaluate the effects of a continuous regional hepatic arterial infusion of human recombinant interleukin-2 (IL-2) in dogs with an infusion pump. Preliminary studies demonstrated the ability to culture canine lymphokine-activated killer (LAK) cells in vitro and a canine LAK cell 15Cr assay was developed with a canine tumor cell line (CTAC) with appropriate controls. An in vitro study of the stability of IL-2 in the pump was done with a bioassay and enzyme-linked immunosorbent assay for IL-2 that demonstrated the stability of IL-2 during a 14-day period at 37 degrees C. Infusions of 300, 600, and 1200 units/kg/hr IL-2 were tested in vivo in dogs. LAK cell and natural killer cell activity, blood counts, and hepatic and renal function were monitored for 1 month. No significant natural killer or LAK response or toxicity was found at the 300 unit/kg/hr level. Infusion of 600 units/kg/hr was associated with a significant increase of the cytotoxic activity of peripheral blood lymphocytes after 3 weeks of treatment. At the 1200 unit/kg/hr level, increased activity occurred at 1 week and thereafter. The only significant toxicity was a 15% increase in body weight occurring during the infusion of 1200 units/kg/hr. Results of renal and hepatic function studies remained normal except for a slight elevation of transaminase levels after 4 weeks of 1200 units/kg/hr. A significant rise in eosinophil count was noted at each dosage level. Results of autopsies were unremarkable. These data demonstrate that continuous hepatic arterial regional infusion with relatively low doses of IL-2 is able to stimulate a sustained in vivo peripheral blood LAK cell effect in dogs with the absence of major side effects. These findings suggest that these methods may have both research application in large animals and clinical application in patients with tumors that are responsive to LAK cell lysis.

Adoptive immunotherapy of human pancreatic cancer with lymphokine-activated killer cells and interleukin-2 in a nude mouse model.

A pancreatic cancer cell line was grown in orthotopic and heterotopic positions in young Swiss/NIH nude mice, which were tested with adoptive immunotherapy. Mice were injected with 1 x 10(7) human cancer cells in the subcutaneous tissue and duodenal lobe of the pancreas. The mice were randomly divided into four groups: group IA (LAK + IL-2) (N = 25) received 2 X 10(7) human lymphokine-activated killer (LAK) cells from normal donors by tail vein injection followed by 10,000 units of human recombinant interleukin-2 (IL-2) given intraperitoneally every 12 hours for 28 days; group IB (IL-2) (N = 27) was given the same dose of IL-2 alone; group IC (RPMI-1640) (N = 18) received a placebo consisting of 1 ml of RPMI-1640 intraperitoneally every 12 hours; and group ID (LAK) (N = 14) received 2 X 10(7) LAK cells but no IL-2. Toxicity was significantly higher in group IB, with a mortality rate of 45.5% (10/22 animals) versus a 0% mortality (0/25) in group IA. None of the group IA or IB animals died of pancreatic cancer during the experiment. The animals that did not receive IL-2 died before 28 days in 14.2% of group IC and in 16.7% of group ID. The area under the growth curve of subcutaneous tumors during the course of treatment and the pancreatic tumor weight at the end of treatment were compared in each group. Subcutaneous tumors had a reduced rate of growth in group IA animals compared to all the other treatments. Pancreatic tumor growth was slowed in group IA. The animals treated with IL-2 alone (group IB) showed some slowing of tumor growth that was intermediate between group IA, group IC, and group ID. A similar experiment was done with irradiated (375 rad) mice. Nine nude mice with tumors were treated with LAK + IL-2 (group IIA), eight received IL-2 alone (group IIB), and seven received placebo (group IIC). The antitumor effect of IL-2 alone was not present in the irradiated mice. A highly significant difference persisted between group IIA and all other groups. There was no difference in the histologic characteristics of tumors in control mice and in mice with inhibited tumor growth treated with IL-2 or IL-2 and human LAK cells. These results show that adoptive immunotherapy with human LAK cells and human recombinant IL-2 is effective against human pancreatic cancer growing in nude mice. This effect is independent from antitumor activity from IL-2 administrations alone.

The in vivo distribution of human peripheral blood lymphocytes and lymphokine-activated killer cells adoptively transferred in human pancreatic cancer-bearing nude mice.

In this study we evaluated human pancreatic cancer xenotransplanted into nude mice as a model suitable for adoptive immunotherapy studies. A pancreatic cancer cell line (MIA PaCa-2) was chosen and its growth in nude mice and sensitivity to lysis by human lymphokine-activated killer (LAK) cells were characterized. This line grew in 96% of the cases when young (4- to 6-week-old) Swiss/NIH nude mice were used. The line was highly sensitive to lysis by LAK cells in a standard chromium-51 release assay (67.8%), similarly to other cell lines known to be highly sensitive, such as K562 (75.6%) and the melanoma cell line SU.102 (53.1%). To assess their in vivo distribution, human peripheral blood lymphocytes (PBLs) and LAK cells were adoptively transferred into nude mice after labeling with indium-111 oxine. The results of this study show that adoptively transferred PBLs and LAK cells localize in this heterologous system as they do in autologous systems. PBLs are taken up mostly by the liver and spleen. The percentage of the administered dose of radioactivity taken up corrected by weight (percent dose per gram tissue) is 64.3 +/- 15.6%d/gm (liver) and 43.5 +/- 9.5%d/gm (spleen). LAK cells are taken up by liver (43.2 +/- 5.3%d/gm) and spleen (28.0 +/- 4.9%d/gm) but also localize significantly more than PBLs in other organs such as lungs (12.9 +/- 3.5%d/gm vs 1.4 +/- 0.3%d/gm, p less than 0.01), kidneys (19.1 +/- 2.1%d/gm vs 6.3 +/- 1.5%d/gm, p less than 0.001), and pancreatic tumors growing in orthotopic position (1.93 +/- 0.36%d/gm vs 0.56 +/- 0.06%d/gm, p less than 0.05). When the nude mice are pretreated with human recombinant tumor necrosis factor, localization of LAK cells compared with PBLs is even further enhanced both in tumors implanted in the pancreas (3.1 +/- 0.5%d/gm vs 0.56 +/- 0.06%d/gm, p less than 0.01) and in the subcutis (12.5 +/- 8.3%d/gm vs 0.95 +/- 0.29%d/gm, p less than 0.001).

Neurologic degeneration associated with nitrous oxide anesthesia in patients with vitamin B12 deficiency.

Vitamin B12 (cyanocobalamin) is an integral component of two biochemical reactions in man: the conversion of L-methylmalonyl coenzyme A into succinyl coenzyme A and the formation of methionine by methylation of homocysteine. The transmethylation reaction is essential to DNA synthesis and to the maintenance of the myelin sheath by the methylation of myelin basic protein. Active vitamin B12 contains cobalt in its reduced form (Co+). Nitrous oxide produces irreversible oxidation to the Co++ and Co forms that renders vitamin B12 inactive. Five cases (four from the literature and one new case) are presented in which patients unsuspected of having vitamin B12 deficiency developed subacute combined degeneration of the spinal cord following nitrous oxide anesthesia. Patients with vitamin B12 deficiency are exceedingly sensitive to neurologic deterioration following nitrous oxide anesthesia. If unrecognized, the neurologic deterioration becomes irreversible and may result in death.

Recognition, management, and prevention of Clostridium septicum abscess in immunosuppressed patients.

Spontaneous gas gangrene due to Clostridium septicum is a rapidly progressing disease that usually ends in fatal toxemia. We report three cases of asymptomatic C septicum abscesses to document the clinical course of this entity and to establish guidelines for its prevention and treatment. In contrast to previously reported data, C septicum infections can produce abscesses in solid organs, the retroperitoneum, and the extremities. These lesions often occur in patients with cancer, producing liver abscesses without gas formation that may be misinterpreted as metastatic carcinoma. Symptoms may be minimal or nonspecific before fulminant toxemia. Asymptomatic bacteremia should prompt a search for unsuspected cancer and an abscess. Computed tomography is the diagnostic modality of choice. The treatment consists of surgical débridement of necrotic tissue in concert with an appropriate course of antibiotics. We have found recurrences after adequate débridement and short-term antibiotic therapy, suggesting that prolonged and even lifelong prophylactic oral penicillin G potassium may be necessary to prevent further recurrences.

Melanoma inhibitory activity (MIA), a serological marker of malignant melanoma.

Melanoma inhibitory activity (MIA) was originally identified as an 11 kDa protein secreted from malignant melanoma cells. We have shown that MIA is strongly expressed in melanoma and melanoma cell lines but not in melanocytes and normal skin. We also observed that MIA mRNA expression correlates with progressive malignancy of melanocytic tumors. Measuring MIA in serum or plasma by a sensitive and quantitative ELISA and investigating the potential of MIA serum levels as a novel marker for malignant melanomas showed that the protein can be used to monitor therapy and follow-up. The present study measured the variations in blood concentrations of MIA in 84 patients with stage II-IV melanoma by ELISA. Patients treated with repeated injections of a polyvalent melanoma vaccine (PMV), interferon-alpha-2b (IFN-alpha 2b) or interleukin-2 (IL-2) were followed during treatment duration. Before treatment, patients treated with PMV or IFN-alpha 2b had comparable low MIA concentrations, whereas most IL-2-treated patients had higher MIA levels. At the end of treatment, MIA concentrations were higher in patients with progressive disease (PD) than in patients with no clinical evidence of melanoma (NPD) for PMV, IFN-alpha 2b or IL-2 therapy (3.7 +/- 0.2 vs 11.5 +/- 5.4 ng/ml, 3.8 +/- 0.2 vs 8.3 +/- 1.7 ng/ml, and 2.3 +/- 0.7 vs 20.2 +/- 7.4 ng/ml, respectively, p < 0.05). In contrast to the stable MIA concentrations measured in NPD patients, significant increase in MIA levels were observed in PD patients over time regardless of treatment. For PMV- and IFN-alpha 2b-treated patients, a rise in MIA levels occurred significantly earlier than clinical diagnosis of melanoma recurrence. In conclusion, our data suggest that quantitation of MIA serum levels may be used for detection of both clinically apparent and non-apparent metastatic melanoma disease and for monitoring therapy.

Absorbable mesh aids in development of discrete, tissue-engineered constructs.

Absorbable mesh was investigated as a potential containment material in which to house discrete, small, tissue-engineered constructs. The mesh was fashioned into bags of varying shapes and consistent volumes. Cells were cultivated on porous, collagen beads, and the tissue constructs were placed into the bags. The mechanical integrity of the bags and feasibility of the design was tested in vitro. The bags successfully maintained their integrity as the cells developed on the collagen matrices. Furthermore, their porosity allowed access of nutrients and waste products to and from the developing tissue. Having demonstrated feasibility of processing, the next step is to optimize the cell culture specifications and materials design.

Angiogenic and immune parameters during recombinant interferon-alpha2b adjuvant treatment in patients with melanoma.

As an adjuvant therapy for patients with high risk of recurrent melanoma, high-dose interferon (IFN)-alpha2b therapy has been shown to have some efficacy. We examined 22 patients with resected melanoma who were treated with repeated injections of recombinant IFN-alpha2b during the treatment. Both angiogenic and immune parameters were measured. White blood cells (WBCs) and lymphocyte numbers, lymphocyte subpopulations, serum concentrations of IFN-alpha and anti-IFN-alpha antibodies, and the serum vascular endothelial growth factor (VEGF), interleukin (IL)-8, and basis fibroblast growth factor (bFGF) concentrations were determined over time in resected, recurrence-free patients with American Joint Committee on Cancer (AJCC) stage III melanoma with one or less (LN+ < or = 1, n = 7) or more than one (LN+ > 1, n = 8) lymph nodes involved, and AJCC stage IV resected disease (n = 7). Follow-up and recurrence-free intervals were longer in stage III (LN+ < or = 1) patients compared with stage IV patients (P < 0.05). The number of WBCs and lymphocytes decreased during the treatment for all patient groups (P < 0.001). In addition, percentages of CD8 and CD20 were higher in stage IV patients than in stage III (LN+ > 1) and stage III (LN+ < or = 1) patients at the beginning of therapy (P < 0.05). A significant increase in the percentage of CD20+ cells, mostly B lymphocytes, was observed in the stage III (LN+ > 1) and stage III (LN+ < or = 1) patients over time but not in stage IV patients (P < 0.001). Low IL-8 and bFGF concentrations at the beginning of therapy were associated with significantly longer recurrence-free survival (P < 0.05). These results warrant a larger trial to determine if the differences observed in patients before treatment can provide prognostic markers in patients receiving IFN-alpha2b therapy.

Melanoma-inhibitory activity protein concentrations in blood of melanoma patients treated with immunotherapy.

Melanoma inhibitory activity protein (MIA) has been detected in patients with advanced melanoma. The present study measured the variations in blood concentrations of MIA in 84 patients with AJCC stage II to IV melanoma by ELISA. Patients treated with repeated injections of a polyvalent melanoma vaccine (PMV), interferon-alpha-2b (IFN-alpha2b), or interleukin-2 (IL-2) were followed during treatment duration. Before treatment, patients treated with PMV or IFN-alpha2b had comparable low MIA concentrations, whereas most IL-2-treated patients had higher MIA levels. At the end of treatment, MIA concentrations were higher in patients with progressive disease (PD) than in patients with no clinical evidence of melanoma (NPD) for PMV, IFN-alpha2b, or IL-2 therapy (3.7+/-0.2 vs. 11.5+/-5.4 ng/ml, 3.8+/-0.2 vs. 8.3+/-1.7 ng/ml, and 2.3+/-0.7 vs. 20.2+/-7.4 ng/ml, respectively, P<0.05). In contrast to stable MIA concentrations measured in NPD patients, significant increase in MIA levels were observed in PD patients over time regardless of treatment (P<0.05). In 20 of the 27 patients who had melanoma recurrence or progression, MIA concentrations were above 4.5 ng/ml. Finally, in these 20 patients, MIA concentrations above 4.5 ng/ml were observed prior to clinical evidence of progression (P<0.01).

Development of technologies aiding large-tissue engineering.

There are many clinical situations in which a large tissue mass is required to replace tissue lost to surgical resection (e.g., mastectomy). It is possible that autologous cell transplantation on biodegradable polymer matrices may provide a new therapy to engineer large tissue which can be used to treat these patients. A number of challenges must be met to engineer a large soft tissue mass. These include the design of (1) a structural framework to maintain a space for tissue development, (2) a space-filling matrix which provides for localization of transplanted cells, and (3) a strategy to enhance vascularization of the forming tissue. In this paper we provide an overview of several technologies which are under development to address these issues. Specifically, support matrices to maintain a space for tissue development have been fabricated from polymers of lactide and glycolide. The ability of these structures to resist compressive forces was regulated by the ratio of lactide to glycolide in the polymer. Smooth muscle cell seeding onto polyglycolide fiber-based matrices has been optimized to allow formation of new tissues in vitro and in vivo. Finally, polymer microsphere drug delivery technology is being developed to release vascular endothelial growth factor (VEGF), a potent angiogenic molecule, at the site of tissue formation. This strategy, which combines several different technologies, may ultimately allow for the engineering of large soft tissues.

Intestinal obstruction.

Acute intestinal obstruction is an emergency that frequently requires operative intervention, either immediately or within several days. Because of the difficulty in distinguishing strangulation obstruction from simple obstruction, a philosophy of operating on all patients with intestinal obstruction has been advocated by many physicians. On the other hand, the approach of attempting tube decompression on most patients rather than immediate operation has also been advocated. Both of these approaches are extreme and somewhat simplistic in dealing with a problem that often is complex. Fluid and electrolyte replacement, intestinal tube decompression, and appropriate surgical management are crucial to the successful management of these patients. This text presents a summary of the pathogenesis and various etiologies of intestinal obstruction and offers some diagnostic and treatment guidelines that will assist the clinician in the management of his patients.

Site-directed metastasis: a model to study site variations in treatment response.

A model for the site-directed metastasis of a transplantable rat mammary carcinoma is described. The R3230AC tumor is a moderately well differentiated, phenotypically stable tumor which arose in a Fischer 344 rat and resembles human breast carcinoma in being hormonally sensitive, poorly immunogenic, and sensitive to radiation and chemotherapeutic agents. Metastases with this tumor can be consistently produced at specific sites (lung, liver, or subcutaneous) in a reproducible manner by the methods described in this paper. This model appears ideal for the evaluation of site-specific variation in response to treatment modalities.

Human papilloma virus in melanoma biopsy specimens and its relation to melanoma progression.

OBJECTIVES: To evaluate melanoma biopsy specimens for human papilloma virus (HPV) and determine the relation between the presence of HPV, in vitro growth, and clinical progression of melanoma in the patients from whom the biopsy specimens were derived. SUMMARY BACKGROUND DATA: Ultraviolet radiation from sun exposure appears to be the primary causal agent in the development of cutaneous melanoma. However, other agents, including HPV, as observed in different epithelial carcinomas, may also play a role in melanoma development and progression. METHODS: Twelve melanoma biopsy specimens obtained from 12 patients with AJCC stage III and IV melanoma were stained with antibodies against gp-100 (HMB-45) and S-100 protein to confirm melanoma diagnosis and with a polyclonal HPV antibody. After mechanical dissociation, the melanoma specimen cells' ability to grow in vitro was assessed. Patients were evaluated for melanoma progression with physical examination, complete blood count, and liver function tests every 3 months and a chest radiograph every 6 months. RESULTS: All biopsy specimens were positive for S-100, and nine (75%) were positive for gp-100. Seven of 12 (58%) were positive for HPV by immunohistochemistry. In vitro, none of the HPV-negative tumor cells grew from the tumor biopsies, whereas five of seven (71%) of the HPV-positive melanoma tumor cells grew very well. All patients with HPV-positive tumor cells had recurrences and died of melanoma progression, whereas four of five (80%) patients with HPV-negative tumor cells remained alive and without melanoma recurrence. CONCLUSIONS: The presence of HPV was found in 58% of the biopsy specimens obtained from patients with stage III and IV melanoma and correlated with rapid melanoma progression. HPV may serve as a cofactor in the development of melanoma and may modulate a more aggressive phenotype in HPV-containing melanoma cells.

Evaluation of a breast cyst fluid protein detectable in the plasma of breast carcinoma patients.

A radioimmunoassay has been developed for one of the major proteins isolated from human breast cyst fluid. Immunologically this protein is identical to a protein present in both human milk and saliva. Ninety-two normal women had plasma levels of this protein below 100 ng/ml (range 7-81 ng/ml; mean 31 ng/ml), and 85% had plasma levels below 50 ng/ml. More than one-half of the women with active gross cystic disease of the breast had plasma levels above 50 ng/ml. None of these women, however, had plasma levels above 150 ng/ml. A significant percentage of women with advanced breast carcinoma have been found to have plasma concentrations of this protein greater than 150 ng/ml.