Sensitizing T-lymphocytes for adoptive immunotherapy by vaccination with wild-type or cytokine gene-transduced melanoma.

BACKGROUND: For the relatively nonimmunogenic B16-F10 murine melanoma, it has been found that genetically engineered expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin (IL)-2, IL-4, or interferon-gamma (IFN-gamma) resulted in a vaccine that could induce resistance to rechallenge. Because T cells from lymph nodes draining the sites of some progressive tumors can mediate tumor regression after in vitro activation, it seemed possible that even apparently nonimmunogenic melanoma cells might induce similar preeffector cells in the vaccine-draining lymph nodes (DLNs). METHODS: C57BL/6 mice were vaccinated with B16-F10 cells that were either unmodified or genetically modified to produce IL-2, IL-4, GM-CSF, or IFN-gamma. DLNs were harvested 10 days after vaccination for adoptive immunotherapy (AIT). The DLN cells were activated with bryostatin 1 and ionomycin (B/I), expanded for 10 days in culture, and transferred to mice with 3-day pulmonary metastases. Pulmonary nodules were counted 14 days after AIT. RESULTS: Adoptive transfer of expanded DLN lymphocytes sensitized by inoculation of WT B16-F10, or IL-4, GM-CSF, or IFN-gamma expressing cells significantly reduced pulmonary metastases. Despite the spontaneous regression of IL-2-transduced B16-F10 tumors, DLN from mice inoculated with IL-2 producing B16 cells had little or no antitumor activity. CONCLUSIONS: B16-F10 vaccination strategies that apparently do not induce systemic immunity can effectively sensitize DLN preeffector cells.

Ophthalmic thermal injuries.

Reflex lid closure often protects the eyes during facial burns. Although corneal burns are uncommon, other ophthalmic injuries occur more frequently. Ophthalmic burns are usually associated with marked facial damage and possible inhalation injury. Failure to recognize and appropriately treat ophthalmic burns can lead to catastrophic sequelae. We performed a 2-year survey of all facial burns in our burn unit. Forty-four patients with thermal facial burns were identified. Sixteen patients had ophthalmic injuries. Corneal injury was detected in 13 per cent (2/16). Intubation was required in 43.75 per cent (7/16) of patients with ophthalmic injuries. Mortality was 25 per cent (4/16). We conclude that patients with facial burns severe enough to cause ophthalmic injuries may be associated with other lethal injuries, and a high index of suspicion should be maintained until all lethal injuries are ruled out. All ophthalmic injuries should be evaluated by an ophthalmologist.

Bryostatin 1 activates splenic lymphocytes and induces sustained depletion of splenocyte protein kinase C activity in vivo after a single intravenous administration.

Bryostatin 1 activates and subsequently down-regulates protein kinase C (PKC) in vitro and has potential use as an immunomodulator and as an anti-cancer agent. Despite extensive examination of its activities in vitro and anti-tumor effects in vivo, previous studies have failed to document that bryostatin 1 modulates total cellular PKC activity in tumor or normal tissues when administered in vivo. After a single bolus injection of bryostatin 1 (1.0 microgram) in normal C57BI/6 mice, blood was drawn at various intervals and assayed for bryostatin 1 levels. In addition, spleens from bryostatin-treated mice were harvested 10 min to 10 days after treatment, weighed and analyzed for cell numbers, PKC activity and cell surface phenotypes. Bryostatin 1 levels in plasma rose rapidly, reaching peak levels of 56.5 nM less than 1 min after injection, and then declined to undetectable levels by 1 h. A similar pattern was observed when bryostatin 1 was incubated with leukemia cells in vitro, raising the possibility that the rapid fall in plasma levels results from intracellular uptake and binding. Bryostatin 1 induced marked depletion of total splenocyte PKC activity (as much as 69% relative to control values) at 24-96 h after drug administration, but not at earlier times (i.e. 1 h). A single injection of bryostatin 1 also induced expression of the T cell activation marker CD69, leading to positivity in 53% of cells at 3-24 h versus 11% in control mice, and resulted in marked splenomegaly, associated with increased numbers of nucleated cells at 48-96 h. Together, these studies demonstrate that despite rapid disappearance of the drug from plasma, a single i.v. dose of bryostatin 1 exhibits significant and sustained effects on normal murine spleen cells, including early lymphocyte activation, prolonged depletion of PKC activity, splenocyte proliferation and splenomegaly. These findings may have implications for attempts to understand the in vivo effects of bryostatin 1 in normal host tissues.

Complications of mastectomy and their relationship to biopsy technique.

BACKGROUND: Wound complication rates after mastectomy are associated with several factors, but little information is available correlating biopsy technique with the development of postmastectomy wound complications. Fine-needle aspiration (FNA) biopsy is an accurate method to establish a diagnosis, but it is unknown whether this approach has an impact on complications after mastectomy. METHODS: Charts of 283 patients undergoing 289 mastectomies were reviewed to investigate any association between biopsy technique and postmastectomy complications. RESULTS: The diagnosis of breast cancer was made by FNA biopsy in 50%, open biopsy in 49.7%, and core needle biopsy in 0.3%. The overall wound infection rate was 5.3% (14 of 266), but only 1.6% when FNA biopsy was used compared with 6.9% with open biopsy (p = 0.06). Among 43 patients undergoing breast reconstruction concomitantly with mastectomy, the infection rate was 7.1% (0% after FNA, 12% after open biopsy). Neither the development of a postoperative seroma (9.8%) nor skin flap necrosis (5.6%) was influenced by the biopsy technique used. CONCLUSIONS: These data suggest that wound infections after mastectomy may be reduced when the diagnosis of breast cancer is established by FNA biopsy.

Adoptive transfer of bryostatin-activated tumor-sensitized lymphocytes prevents or destroys tumor metastases without expansion in vitro.

Because the requirement for long-term cell culture can make adoptive cellular immunotherapy cumbersome, experiments were designed to determine whether smaller numbers of tumor-sensitized T cells activated briefly with bryostatin 1 and ionomycin (B/I) could be returned immediately to recipient mice without in vitro expansion and still have an anti-tumor effect in vivo. Popliteal tumor-draining lymph nodes (DLNs) from mice bearing progressive MCA-105 and MCA-203 footpad sarcomas were harvested and treated for 18 h with B/I. These cells were then washed and transferred immediately to naive C57B1/6 mice. In some experiments, these mice were irradiated (500 rads) before adoptive transfer and were given interleukin-2 (IL-2, 7,500 IU i.p., b.i.d. for 3 days) after receiving the activated lymphocytes. Recipient mice were challenged with sarcoma cells (4 x 10(5) i.v.) 6 to 32 days after receiving the activated lymphocytes. Mice receiving 10(6) B/I-activated lymphocytes before tumor challenge had significantly fewer metastases than did controls. This protective effect did not require exogenous IL-2 or host irradiation. Using Thy-1 congenic donors, it was shown that B/I-activated T cells expanded in recipients when IL-2 was also given, and these cells were a prominent component (15% of total cells) in the infiltrates found in the lungs of mice 7 days after i.v. tumor challenge. Combining these B/I-"pulsed" cells with cyclophosphamide (CYP) and IL-2 to treat mice with established (3-day) metastases resulted in significant reduction in pulmonary nodules, with complete regression in many of the treated mice, which was rarely seen with CYP alone or with CYP + IL-2. Thus, adoptive transfer of tumor-sensitized, B/I-activated DLN cells confers protection against i.v. tumor challenge, without prior in vitro expansion of the effector cells. Phenotyping studies demonstrate that donor cells activated with B/I do expand in recipient mice after adoptive transfer and can move to sites of tumor. Moreover, these cells can mediate a therapeutic effect on established tumor metastases, when combined with chemotherapy.

Missed pathology following laparoscopic cholecystectomy: a cause for concern?

Although many have recorded the incidence of complications after laparoscopic cholecystectomy, few have discussed the possibility of missing intra-abdominal pathology after this procedure. We have evaluated the first two years, September 1990-September 1992, of laparoscopic cholecystectomy in our community. Readmissions within 10 months of the original surgery with another diagnosis similar to gallbladder disease were considered "missed pathology" at the original surgical procedure. For the first 12-month period, 465 laparoscopic cholecystectomies were performed. Seventy-seven patients were readmitted, with 13 of these patients having other intra-abdominal pathology. These readmissions were for carcinoma (6), inflammatory bowel disease (2), diverticular disease, esophageal varices, and appendicitis. In the second year 429 laparoscopic cholecystectomies were performed; 59 patients were readmitted, with 10 of these patients having other intra-abdominal pathology. These readmissions were for carcinoma (3), inflammatory bowel disease (2), strongyloides, peptic ulcer disease, and abdominal pain of unknown etiology (3). Although intra-abdominal pathology was found in only 2%-5% of all patients having surgery for gallbladder disease, of the patients who were readmitted for "missed pathology," 46% the first year and 30% the second year were readmitted for carcinomas. Several other diseases were found in patients whose symptoms mimicked gallbladder disease. It is therefore possible that in the zeal to perform a new procedure, other diagnoses may be overlooked.