A pilot study of autologous cancer cell vaccination and cellular immunotherapy using anti-CD3 stimulated lymphocytes in patients with recurrent grade III/IV astrocytoma.

The study objectives were to determine; (1) whether activated T cells could be generated from peripheral blood of patients immunized with their own cancer cells, (2) whether adoptive transfer of the activated T cells to patients had toxic effects and (3) whether the infused cells produced clinical responses. Study patients had recurrent, surgically accessible grade III/IV astrocytomas. The patients were tapered off steroids after total surgical resection and immunized with autologous cancer cells plus Bacillus, Calmette and Guerin (BCG). Peripheral blood mononuclear cells were activated with anti-CD3, expanded with interleukin-2 (IL-2) and reinfused to patients. The number of activated T cells that was given back to patients varied between 10(10) and 10(11). Side effects that were observed following immunization and adoptive cell transfer included mainly transient flu-like symptoms. One patient's tumor partially regressed, but there was no effect on survival. Two other patients' tumors regressed, and the patients are apparently disease-free more than 5 and 4 years later. The other six patients' tumors were apparently unaffected by the treatment. Patient age, tumor grade and CD4/CD8 composition of infused cells were positively correlated with clinical responses. Cellular immunotherapy is feasible and is associated with minimal toxicity. Additional appropriately controlled studies will be required to determine whether cellular immunotherapy could be used as a treatment for central nervous system malignancy. Additional studies also will be required to determine the underlying immunological mechanisms.

Immune rejection of intracerebral gliomas using lymphocytes from glioma-bearing rats.

Naturally occurring malignancies do not induce immune responses against cancer antigens. Is the lack of an immune response caused by an antigen presentation defect or by induced antigen-specific immune suppression? The current study was performed to determine whether a progressing intracerebral malignancy affects production of peripheral autologous glioma antigen-specific immune responses. Peripheral immunization of both glioma-bearing and non-glioma-bearing animals with cancer cells and adjuvant generated similar levels of glioma antigen-specific cytotoxic T lymphocyte activity. However, immune cell populations from glioma bearers were significantly less efficient than immune cell populations from non-cancer bearers in their ability to reject progressing intracerebral tumors. A variety of manipulations designed to reduce nonspecific immune suppression in vivo and in vitro had no effect on the in vivo efficacy of the activated T-lymphocyte populations. The presence of progressing tumors appeared to augment rather than suppress cancer antigen-specific responses, leading to the speculation that reduced efficacy was caused not by generalized immune suppression but rather by a reduction in the number of immune effector cells by either clonal anergy or clonal deletion. Most importantly, the data demonstrated that, despite decreased in vivo efficacy, immune effector cells capable of rejecting an intracerebral malignancy could be generated from cancerous hosts.

Autologous tumor cell vaccination combined with adoptive cellular immunotherapy in patients with grade III/IV astrocytoma.

Brain tumors are highly resistant to treatment. Their diffuse infiltrative nature and the relative inaccessibility of the brain to blood and lymph are barriers to surgical and cytotoxic treatments alike. Preclinical animal studies demonstrated that intravenously administered tumor antigen-specific T lymphocytes will reject tumors growing in the brain. Specifically activated effector T lymphocytes may be generated by in vivo immunization followed by restimulation of antigen-primed T cells with autologous tumor cells in vitro. In order to apply these findings to humans, feasibility studies of combined active immunization and specific adoptive cellular immunotherapy were performed on fifteen patients with recurrent astrocytoma. The objective was to determine whether; 1) T cells could be grown from peripheral blood of patients immunized with autologous tumor cells, and 2) whether stimulated cells could be safely readministered to patients. Patients were immunized with a combination of their own irradiated tumor cells and Bacillus of Calmette and Guerin. Two weeks later a mononuclear cell-rich fraction of blood was obtained by leukapheresis. Mononuclear cells were cultured with irradiated autologous tumor cells and interleukin-2. Selective expansion of CD4+ and CD8+ T lymphocytes occurred. Intravenous transfer of stimulated cells to the fifteen patients on twenty-four separate occasions with or without systemic administration of interleukin-2 was tolerated with limited toxicity. The studies established the feasibility of conducting controlled studies of the anti-tumor effects of tumor antigen-specific cellular immunotherapy.

Continuous intraoperative electromyographic recording during spinal surgery.

One hundred fifty patients underwent spinal surgery for radiculopathy; of these, 120 underwent lumbar surgery and 30 had cervical operations. All of the surgeries were performed to alleviate symptoms due to disc herniation, spondylosis, or both. During the surgical procedures continuous intraoperative electromyograph recordings were taken from the muscle corresponding to the involved nerve root. In baseline recordings taken in the operating room 10 minutes before lumbar surgery, electrical discharge or firing was recorded from the muscle in 18% (22 of 120 patients) of the cases. Once the nerve was decompressed, muscle firing ceased. Electrical discharges were produced with regularity on nerve root retraction. This study concludes that continuous electromyograph monitoring can be accomplished easily and yields valuable information that indicates when the nerve root is adequately decompressed or when undue retraction is exerted on the root.

Primary meningeal extraosseous Ewing's sarcoma: case report.

A 25-year-old man presented with a suspected right-sided subdural hematoma after a skiing accident. A large hemorrhagic mass was found and was evacuated. Histological studies demonstrated a highly cellular neoplasm with extensive hemorrhage. Further histological, immunohistochemical (including staining for Ewing's sarcoma cell surface antigen), and ultrastructural analyses of the tumor were consistent with Ewing's sarcoma. Search for other foci of this neoplasm by bone scan, full body computed tomographic scans, magnetic resonance imaging scans of the spine, and a bone marrow aspiration with biopsy failed to detect any soft tissue or bony involvement outside the cranium. This case appears to represent the first report of a primary extraosseous Ewing's sarcoma occupying the cranial subdural area.

Generation of cytotoxic immune responses during the progression of a rat glioma.

Cytotoxic T lymphocytes specific for tumor-associated antigens are produced by exposing animals to tumor cells and stimulating lymphocytes from animals immunized in vitro with tumor cells and small amounts of interleukin-2 (IL-2). This study was designed to determine whether a fast-growing immunogenic avian sarcoma virus-induced glioma produces primed cytotoxic T lymphocyte precursors during its progression. Lymphocytes from intracerebral glioma-bearing rats generally failed to proliferate in vitro in response to immunization with tumor cells and IL-2 and, when proliferative responses were observed, the lymphocytes were not cytotoxic for glioma cells. However, when the same tumor was growing subcutaneously, lymphocytes proliferated and exhibited glioma-specific cytotoxicity when stimulated in vitro with autologous tumor cells and IL-2. Subcutaneous immunization of intracerebral glioma-bearing rats with tumor cells and adjuvant induced strong cytotoxic T lymphocyte responses. The results demonstrated that, while intracerebral tumor progression itself does not induce an anti-glioma immune response, immune responses to tumor-associated antigens may be induced by systemic immunization of tumor-bearing animals. The results suggest that the immunogenicity of brain tumors is masked by the immunologically privileged status of the brain, not by the induction of generalized immune suppression during tumor progression.

Generation of cellular immune responses against a glioma-associated antigen(s).

The study demonstrated that RT2, a highly malignant anaplastic glioma, expresses antigens that make it susceptible to in vivo adoptive immunotherapy with cytotoxic T lymphocyte-containing immune cell populations. Rats were immunized with irradiated RT2 tumor cells and the adjuvant C. parvum. Lymphocytes from immunized rats were restimulated in vitro with irradiated RT2 tumor cells plus interleukin-2 (IL-2). The cells that proliferated and differentiated in vitro effectively killed RT2, but only low levels of cytotoxicity were observed against other histopathologically related and unrelated, syngeneic and allogeneic target cells. Adoptive transfer of immune cells combined with a 5-day course of systemic IL-2 produced specific regression of brain tumors growing as lung microfoci.

Antitumor activity against established intracerebral gliomas exhibited by cytotoxic T lymphocytes, but not by lymphokine-activated killer cells.

Specific immune responses against malignant brain tumors have been difficult to demonstrate. Moreover, immunotherapy has met with little success, despite using lymphocytes with high levels of cytotoxicity against brain tumor cells. Lymphokine-activated killer (LAK) cells that nonspecifically kill brain tumor cells are produced by stimulating resting precursors with high concentrations of interleukin-2 (IL-2). Cytotoxic T lymphocytes that specifically kill brain tumor cells are produced by stimulating antigen receptor-positive immune-cell precursors with tumor cells. In an attempt to gain insight into immune cell function against brain tumors, the present study compared the in vitro and in vivo activities of LAK cells and cytotoxic T lymphocytes produced against RT2, a fast-growing rat glioma cell line. Lymphokine-activated killer cells were produced by stimulating normal rat spleen cells with 1000 units of IL-2, and RT2-specific cytotoxic T lymphocytes were produced by priming them in vivo with RT2 and Corynebacterium parvum and restimulating primed spleen cells with RT2 in vitro. Lymphokine-activated killer cells were highly cytotoxic for a panel of syngeneic and allogeneic brain tumor and non-brain tumor target cells, including RT2, as measured in a 4-hour 51Cr release assay. Cytotoxic T lymphocytes were highly cytotoxic only for syngeneic brain tumor target cells. Lymphokine-activated killer cells and cytotoxic T lymphocytes were tested for in vivo antitumor activity against intracerebral RT2 by intravenous adoptive transfer of activated lymphocytes. Untreated rats died in approximately 2 weeks. Lymphokine-activated killer cells plus IL-2 failed to affect survival when treatment was initiated as early as 1 day following tumor inoculation. Cytotoxic T lymphocytes and IL-2 administered as late as Day 5 rejected progressing intracerebral tumor. Thus, although both cytotoxic T lymphocytes and LAK cells exhibited high levels of in vitro killing of glioma cells, only cytotoxic T lymphocytes rejected progressing intracerebral tumors.

Successful treatment of a malignant rat glioma with cytotoxic T lymphocytes.

Brain tumors are highly resistant to therapy. Their diffuse infiltrative nature and the relative inaccessibility of brain tissue to blood and lymph are barriers to surgical and cytotoxic treatments alike. The purpose of this study was to produce immune cells specifically reactive with an anaplastic rat glioma (RT2) and determine whether those cells could affect tumor progression in the brain. RT2-specific cytotoxic cells were prepared by priming rats in vivo with RT2 tumor cells and Corynebacterium parvum and stimulating the primed lymphocytes in vitro with irradiated RT2 tumor cells and interleukin-2 (IL-2). Cultured cells exhibited a high level of cytotoxicity against RT2, but not C6 (an allogeneic glioma), 3M2N (a syngeneic mammary tumor), or CSE (a syngeneic fibrosarcoma) tumor cells. To generate a model for therapy, rats were injected intracerebrally with RT2, generating progressing brain tumors, which killed untreated rats in approximately 2 weeks. To test the therapeutic potential of the effector cells, tumor-bearing rats were treated by intravenous injection of lymphocytes on Day 5 of tumor growth. Treated rats also received a 5-day course of systemic IL-2 beginning on Day 5. Treatment with IL-2 alone, RT2-primed spleen cells, or RT2-primed spleen cells stimulated in vitro with C6 did not affect rat survival. However, tumor-bearing rats treated with RT2-stimulated lymphocytes exhibited increased survival or were cured. Systemic IL-2 was an essential adjunct, because survival was not affected by treatment with effector cells alone. Therapy initiated on Day 8 of tumor progression lacked effect on survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of cytotoxic immune responses against a rat glioma by in vivo priming and secondary in vitro stimulation with tumor cells.

Cytotoxic T lymphocyte (CTL) responses to most antigens are generated by in vivo priming and secondary stimulation with antigen in vitro. The present studies were designed to determine whether that strategy could be used to stimulate development of CTL against brain tumors. Rats were primed with one of two tumors, RT2, an astrocytoma, or 9L, a gliosarcoma, and Corynebacterium parvum. Spleen cells from primed rats were stimulated with tumor cells and interleukin-2 in vitro to generate CTL. CTL generated against RT2 killed RT2 and 9L, but not allogeneic or histopathologically unrelated tumor cells, suggesting that the killing was brain tumor-specific and major histocompatibility complex gene product-restricted. Similar results were obtained with rats primed and secondarily stimulated with 9L. Specific cytotoxic cells only developed when syngeneic brain tumor cells were used for both priming and secondary stimulation. The cytotoxic cell populations were composed of OX-19+ T cells with a mixed CD4/CD8 phenotype. Controls consisting of spleen cells from unprimed or primed rats tested before culture exhibited low levels of cytotoxicity against brain tumor targets. Culturing unprimed or primed cells with interleukin-2 alone stimulated cell proliferation, but the cells that grew out exhibited only low levels of cytotoxicity for brain tumor cells. Cell populations exhibited consistent cytotoxicity against natural killer cell targets. None of the cell populations killed lymphokine-activated killer cell targets. The results demonstrated that brain tumor-specific CTL could be produced by priming in vivo followed by secondary stimulation with brain tumor cells in vitro. The results further demonstrated that RT2 and 9L share antigens that both induce and serve as target structures for specific cytotoxic cells.

A cerebrospinal fluid glucose biosensor for diabetes mellitus.

A cerebrospinal fluid (CSF) glucose biosensor is introduced. The biosensor is a polarimeter that measures the rotation of plane polarized light proportional to glucose concentration. Preliminary in vitro studies revealed a linear response with good sensitivity over a range of glucose solutions (0-400 mg/dl). Anesthetized, adult dogs underwent intravenous glucose loading, and these preliminary in vivo studies resulted in good correlation (r = 0.98) between CSF polarimeter readings and CSF glucose by laboratory assay. This in vivo correlation suggests that both mutarotation of glucose anomer and changes from other optically active substances present in CSF are either negligible or constant over the range of glucose concentrations studied. The CSF polarimeter showed a significant rise soon after the intravenous loading of glucose (1-30 min) but a longer lag time (45-60 min) between the peak blood glucose and peak CSF polarimeter reading. This preliminary work extends, to the CSF, the concept of measuring optical rotation.

Osteomyelitis of the skull base.

Three cases of osteomyelitis of the skull base with associated problems in diagnosis and therapy are discussed. Patients with atypical skull base osteomyelitis are difficult to diagnose as they have no ear abnormalities, but they often develop multiple cranial nerve deficits mimicking symptoms of a posterior fossa mass. We conclude that computed tomographic scans, magnetic resonance imaging studies, bone scans indium-labeled white blood cell scans, and gallium scans are useful in making the diagnosis. A biopsy of the bony lesion often is needed to identify the causative organism and to rule out a tumor. Intravenously administered antibiotics are the mainstay of therapy and should be continued until 1 week after the gallium scan shows no abnormalities. Follow-up gallium scans then are done at 1 week and 3 months after the cessation of antibiotic therapy to search for a recurrence.

Hematocrit monitor.

The purpose of this study is to determine experimentally the optimal incident visible wavelength and light detector angle that yields the maximal change in optical density between an arterial or venous Hct of 20% and 40%. A universal monochromator allows incident wave-lengths in 8-nm increments over the visible range to be selected for the incident beam and a motorized shuttle allows the two samples (Hct 20% and 40%) to be placed reproducibly between the incident beam and photodiode detector. Runs performed with the light detector at a 30 degree, 90 degree, and 180 degree angle from the incident light beam revealed the greatest change in optical density between an Hct of 20% and 40% to occur at 624 nm and 90 degrees independent of sample cell configuration, light source profile, or light detector profile. This experimentally determined optimal wavelength and angle are utilized in a fiberoptic biosensor to monitor Hct successfully in dogs undergoing surgical procedures.

Experimental study of chronic ambulatory peritoneal dialysis. I. Inhibition of protein and amino acid losses by single amino acids.

A successful dog model of the continuous ambulatory peritoneal dialysis patient was developed. These preparations were employed in initial studies of the effects of single amino acid-containing dialysis solutions on the losses of protein and amino acids into the dialysate. A decrease of about 40% in the loss of total amino acids into the dialysate was observed when DL-serine-containing dialysis solutions were employed. The addition of DL-serine, L-lysine, or DL-alanine to the dialysis solutions diminished protein loss into the dialysate by 27-55%. DL-Glutamic acid and DL-aspartic acid were ineffective in this regard.

Effects of verapamil on renin and aldosterone in the dog and rat.

The purpose of this study was to evaluate the effects of calcium channel blockade on renin secretion and plasma aldosterone. Verapamil infusion (0.004 mg X kg-1 X min-1) into the renal artery of uninephrectomized dogs with an intact kidney resulted in significant increases (P less than 0.05) in renal blood flow, creatinine clearance, and the excreted fractions of Na+ and Cl-; renin secretion decreased (P less than 0.05) and plasma aldosterone did not change. Conversely, renal arterial infusion of verapamil in dogs with nonfiltering, denervated, papaverine-treated kidneys resulted in no change in renal blood flow and a significant increase (P less than 0.05) in renin secretion. In the rat, compared with untreated control animals, dietary verapamil (12.5 mg X kg-1 X day-1) did not effect plasma renin activity but significantly suppressed plasma aldosterone (P less than 0.001) in animals on NaCl-restricted [14.7 +/- 5.4 vs. 41.6 +/- 7.8 ng/dl (SE)] and NaCl-free [103.7 +/- 7.5 vs. 156.7 +/- 18.7 ng/dl (SE)] diets. In addition, verapamil suppressed (P less than 0.0003) plasma corticosterone [16.1 +/- 5.4 vs. 52.8 +/- 7.1 microgram/dl (SE)]. Thus, acute but not chronic verapamil administration stimulates renin release in the nonfiltering kidney, and chronic verapamil inhibits adrenal mineralocorticoid and glucocorticoid secretion. The disparate effects of verapamil on renin secretion from intact and nonfiltering kidneys may be due to actions of the Ca2+ channel blocker on renal hemodynamic and/or renal tubular mechanisms in the intact kidney that mask a direct effect of verapamil on renin-secreting cells.

Cerebral vascular response to moderate blood loss: modification by hypertension.

To study the effect of non-hypotensive hemorrhage on cerebral blood flow in normo- and hypertensive states, chloralose anesthetized cats were subjected to graded blood loss (5 ml/kg) every 30 min. Cerebral blood flow was measured using radiolabelled microspheres or H2 clearance. Hypertension was produced by infusion of phenylephrine to a diastolic blood pressure of 100 mm Hg. Control animals suffered no net blood loss. PCO2 was between 28 and 32 mm Hg for all groups over the entire experiment. In normotensive cats, cerebral blood flow increased following withdrawal of 10 ml/kg of blood. In hypertensive cats, cerebral blood flow increased after withdrawal of 20 ml/kg of blood. These findings were consistent for all brain regions examined. Animals without blood loss, whether normo- or hypertensive showed no consistent change in cerebral blood flows. Possible explanations for these findings, particularly neurally mediated responses, are discussed.

cerebellar oligodendroglioma in a child.

Oligodendrogliomas occur primarily in the cerebral hemispheres of adults. A rare case of an oligodendroglioma in the cerebellum of a child is presented. The tendency for oligodendroglioma to metastasize through the cerebrospinal fluid (CSF) is reviewed and emphasized. A recommendation for CSF cytology and possible spinal axis irradiation in the treatment of oligodendrogliomas is made.