Adjuvant therapy of melanoma with interferon-alpha-2b is associated with mania and bipolar syndromes.

BACKGROUND: The use of a high dose regimen of interferon-alpha-2b (IFN) has recently been demonstrated to benefit patients with resected high risk melanoma. The incidence of melanoma is rising rapidly, and the use of this regimen is becoming increasingly common. IFN has been associated with numerous psychiatric side effects. METHODS: The authors describe four melanoma patients treated with adjuvant IFN who developed a manic-depressive syndrome or mood instability with therapy, and they review the literature on mania and the mixed affective syndromes associated with IFN. RESULTS: The authors suggest that IFN may induce a mixed affective instability, and that patients risk developing hypomania or mania as IFN doses fluctuate or as IFN-induced depression is treated with antidepressants alone. Mania is particularly associated with dose reductions or pauses in IFN treatment. The risk of mood fluctuation continues after treatment with IFN stops, and patients should be monitored for 6 months following completion of therapy. Gabapentin appeared effective as monotherapy for acute mania, as an antianxiety agent, as a hypnotic, and as a mood stabilizer in these individual cases. CONCLUSIONS: Mania and mood instability can occur in patients being treated with IFN therapy for melanoma. In this study, gabapentin was an effective mood-stabilizing agent for these patients.

Adjuvant high-dose interferon alfa-2b in patients with high-risk melanoma.

We performed an analysis of toxicity and survival in stage III melanoma patients receiving adjuvant interferon alfa-2b (IFN). This was a retrospective single-arm analysis of 40 patients with stage III melanoma who received (IFN) administered at maximum tolerated doses of 20 mU/m2/day intravenously (i.v.) for 1 month and 10 mU/m2 three times per week subcutaneously (s.c.) for 48 weeks. Toxicity in our series is comparable to that experienced in the Eastern Cooperative Oncology Group (ECOG) 1684 trial, except for higher rates of dose-limiting myelosuppression and hepatotoxicity. All 40 patients experienced constitutional symptoms, but only 14/40 (35%) experienced grade 3 to 4 symptoms. Of the 40 patients, 36 (90%) experienced neurologic symptoms, but only seven (17.5%) experienced grade 3 to 4 neurotoxicity. Two patients stopped treatment because of severe psychiatric symptoms; one patient attempted suicide, and a psychosis developed in another. Thirty-nine (97.5%) patients experienced myelosuppression; 31 (77.5%) developing grade 3 to 4 myelosuppression. Hepatotoxicity was evident in 39 (97.5%) patients, and 26 (65%) experienced grade 3 to 4 hepatotoxicity. Three patients (7.5%) experienced mild renal toxicity. At a median follow-up of 27 months from initiation of therapy, there have been 19 relapses (47.5% disease-free survival [DFS]) and 10 deaths (75% OS) resulting from progression of disease. The DFS compares with the treatment arm in ECOG 1684 at 27 months, but overall survival is higher in our series of patients at the same time point. In a single program setting, IFN can be administered with similar side effects and outcome profiles seen in multi-institutional studies. Modifications in the induction regimen resulted in notably higher hematologic and hepatic toxicities but did not preclude administering further therapy and did not result in increased attrition rate among patients: only nine patients (22.5%) had their treatment stopped as a result of IFN-related toxicity. In comparison, 26% of patients had to have their treatment discontinued because of toxicity in ECOG 1684.

Lymphedema after sentinel lymph node biopsy for cutaneous melanoma: a report of 5 cases.

BACKGROUND: Sentinel lymph node (SLN) biopsy has rapidly become the procedure of choice for assessing the lymph node status of patients with 1992 American Joint Committee on Cancer stages I and II melanoma. The procedure was designed to be less invasive and, therefore, less likely to cause complications than a complete lymph node dissection. To our knowledge, this is the first report in the literature documenting extremity lymphedema following SLN biopsy. OBSERVATION: We report 5 cases of lymphedema after SLN biopsy in patients being routinely followed up after melanoma surgery at the Massachusetts General Hospital Melanoma Center, Boston. Three cases were mild, and 2 were moderate. Potential contributing causes of lymphedema were present in 4 patients and included the transient formation of hematomas and seromas, obesity, the possibility of occult metastatic melanoma, and the proximal extremity location of the primary melanoma excision. Four of the patients underwent an SLN biopsy at our institution. We used the total number of SLN procedures (N = 235) that we have performed to calculate a 1.7% baseline incidence of lymphedema after SLN biopsy. CONCLUSIONS: Sentinel lymph node biopsy can be complicated by mild and moderate degrees of lymphedema, with an incidence of at least 1.7%. Some patients may have contributing causes for lymphedema other than the SLN biopsy, but many of these causes are difficult to modify or avoid.

Detection of microscopic melanoma metastases in sentinel lymph nodes.

BACKGROUND: Sentinel lymph node biopsy following radioisotope labeling is a recently developed, minimally invasive surgical staging procedure used in the management of primary cutaneous malignant melanoma. If histologic analysis reveals melanoma metastasis in the sentinel lymph node, completion lymphadenectomy is performed and adjuvant therapy considered. The routine pathologic assessment of the sentinel lymph node consists of bisecting the lymph node along its long axis and histologic examination of one hematoxylin and eosin-stained section of each cut surface. METHODS: In this study, the authors reexamined 235 sentinel lymph nodes reported as negative for melanoma metastasis following routine histologic examination, from 94 patients with American Joint Committee on Cancer (AJCC) Stage I and II cutaneous melanoma. RESULTS: Deeper sections into the lymph node and immunohistochemical stains with antibodies to S-100, HMB-45, NK1C3, and MART-1 led to the identification of microscopic metastases in 11 sentinel lymph nodes from 11 patients and capsular nevi in 9 sentinel lymph nodes from 8 patients. CONCLUSIONS: Deeper serial sections and immunohistochemical stains detected microscopic metastases in approximately 12% of cases that would be reported as negative for metastasis by routine pathologic analysis. These techniques also allowed for the identification of capsular melanocytic nevi in the sentinel lymph nodes of 9% of patients. [See editorial on pages 551-2, this issue.]

Surgical management of phyllodes tumors.

HYPOTHESIS: Although phyllodes tumors have minimal metastatic potential, we hypothesized that they have a proclivity for local recurrence and should be excised with a wide margin. We reviewed the clinical and radiological appearance of phyllodes tumors and analyzed the role of surgical treatment in their management. DESIGN: Medical records, imaging studies, pathology reports, and interventions were reviewed. SETTING: A large tertiary care teaching hospital. PATIENTS: Between 1980 and 1997, 40 patients with phyllodes tumors were identified through the tumor registry at the Massachusetts General Hospital, Boston. MAIN OUTCOME MEASURES: Surgical resection margins, rates of local recurrence, incidence of distant metastases, and survival. RESULTS: All 40 patients were female, with a mean age of 41 years. Each patient had a palpable mass or a mammographic finding that was indistinguishable from a fibroadenoma on examination. Tumor size ranged from 5 mm to 28 cm. Local recurrence correlated with excision margins (P<.05), but not with tumor grade or size. Local recurrence occurred in 5 patients, each of whom had positive margins or margins less than 1 cm after excision. After reexcision with a 1-cm margin, these individuals remained free of recurrence. One patient developed metastatic disease after total mastectomy and died after chemotherapy. CONCLUSIONS: Phyllodes tumors mimic fibroadenomas and are often excised with close margins. Primary excision or reexcision with a 1-cm margin is recommended. Mastectomy is indicated for patients with large lesions. Lymph node metastases are unusual and occur secondary to necrotic tumor. Chemotherapy is based on guidelines for the treatment of sarcomas, not breast adenocarcinoma.

Outcome of patients with melanoma and histologically negative sentinel lymph nodes.

HYPOTHESIS: Patients with melanoma and histologically negative sentinel lymph nodes identified by lymphatic mapping have a very good prognosis. DESIGN: Cohort study with follow-up information obtained from medical records and telephone interviews. SETTING AND PATIENTS: Of all patients with cutaneous melanoma who underwent intraoperative sentinel lymph node mapping between November 15, 1993, and April 18, 1997, at the Massachusetts General Hospital, Boston, 89 were found to have no evidence of melanoma in their sentinel nodes. Forty-six lesions (51%) were on an extremity and 44 (49%) were of axial location. The median tumor thickness was 1.8 mm (range, 0.36-12.0 mm) and 11 tumors (12%) were ulcerated. INTERVENTIONS: Patients underwent intraoperative sentinel lymph node mapping with lymphazurin and radiolabeled sulfur colloid. Sentinel lymph nodes were analyzed by standard hematoxylin-eosin staining. Only 2 patients received adjuvant therapy following wide excision of the primary lesion. MAIN OUTCOME MEASURES: Site of initial recurrence and time to initial recurrence. RESULTS: The median follow-up for all patients was 23 months (range, 2-54 months). Eleven patients (12%) developed melanoma recurrences, and 78 (88%) patients remain disease free. Regional lymph nodes were the initial site of recurrence in 7 (8%) of 89 patients, and 7 (7%) of 106 mapped basins. Four patients had recurrence without involvement of regional lymph nodes: 2 with distant metastases and 2 with in transit metastases. The median time to recurrence was 12 months (range, 2-35 months). Sentinel lymph nodes were reanalyzed using serial sections and immunoperoxidase stains in 7 patients with recurrence and metastatic melanoma was identified in 3 (43%). CONCLUSIONS: The risk for melanoma recurrence is relatively low in patients with histologically negative sentinel nodes identified by lymphatic mapping. Longer follow-up will improve our understanding of the prognostic value of this procedure.

Multimodality management of Merkel cell carcinoma.

HYPOTHESIS: Merkel cell carcinoma is a rare dermal neuroendocrine carcinoma whose optimal treatment and prognostic factors are poorly defined. We hypothesize that high-risk patients with Merkel cell carcinoma are best treated with multimodality therapy. DESIGN: A retrospective review of all patients (N = 33) with Merkel cell carcinoma treated at the Massachusetts General Hospital from January 1, 1980, to August 24,1997. Median follow-up time was 37 months (range, 6-157 months). PATIENTS: Adequate data for evaluation were available for 31 patients. Male to female distribution was 14 men and 17 women, with a median patient age of 68 years. MAIN OUTCOME MEASURE: Stage at presentation; factors associated with recurrence; and the effects of surgery, radiation therapy (XRT), and chemotherapy on recurrence, salvage, and survival rates. RESULTS: There were 12 extremity, 11 head and neck, and 8 truncal tumors. There were 22 isolated primary tumors, 8 with additional clinically positive lymph nodes, and 1 with distant disease. Therapy was local excision with or without XRT in 19 patients, local resection and lymphadenectomy with or without XRT in 8 patients, and XRT alone in 4 patients with head and neck tumors. Fifteen patients developed recurrences (7 local, 8 nodal, and 10 distant). Median time to recurrence was 8 months (range, 3-48 months). There were 7 tumor-related deaths, 6 of which were associated with truncal lesions (P<.001). No locoregional recurrences occurred in patients with margins of resection of 2 cm or greater or adequate XRT. A multivariate analysis selected truncal location (P = .005) and nodal disease (P = .05) as predictors of mortality. Remission was possible in 5 patients with locoregional and 2 patients with distant recurrences. CONCLUSIONS: Merkel cell carcinoma is an aggressive dermal cancer with frequent nodal metastases; truncal tumors have the worst prognosis. Locoregional recurrence correlates with inadequate margins and lack of XRT, but remission is possible with multimodality therapy.

Surgical management of primary cutaneous melanomas of the hands and feet.

OBJECTIVE: The purpose of the study was to investigate the surgical management of cutaneous melanomas of the hands and feet. SUMMARY BACKGROUND DATA: Prior studies suggest that patients with melanomes > 1-mm thick should be treated with excision with a 2-cm margin and undergo elective lymphadenectomy in selected circumstances. These recommendations are based primarily on data from melanomas of the trunk and extremities. Melanomas of the hands and feet are less common and less well studied. They pose a surgical challenge because primary wound closure often is difficult, and the incidence and management of regional node metastases are unclear. METHODS: Charts of patients with melanomas of the hands or feet treated at the Massachusetts General Hospital between 1980 and 1994 were reviewed retrospectively. Local recurrence rates and the incidence of regional node metastases were analyzed as a function of histology, margin of excision, and microscopic thickness of the melanoma. RESULTS: Data from 116 patients (39 men, 77 women) with melanomas of the hands (n = 26) and feet (n = 90) were evaluated. Pathologic diagnoses were: acral lentiginous melanoma (48 patients); subungual melanoma (13 patients), and skin of dorsum of the hand or foot (n = 55). Digital amputation was required in all 13 patients with subungual melanoma to maintain local control; still, nodal metastases developed in 46% of patients within 1 year. Seventy-one percent of patients with acral lentiginous melanoma presented with lesions > or = 1.5 mm, and nodes or systemic disease or both developed in 56% of patients. Acral lentiginous melanoma lesions < 1.5-mm thick were treated principally by excision with a 1-cm margin; a local recurrence or metastases did not develop in any of the patients. None of the patients with melanomas on the dorsum of the hand or foot < 1.5-mm thick had a local recurrence, but regional or systemic disease developed in > 50%. Local control in patients with lesions > 1.5-mm thick frequently required skin grafting or amputation. The majority of patients with melanomas > or = 1.5 mm in thickness undergoing elective lymph node dissection had histologically positive nodes for melanoma. CONCLUSIONS: Melanomas of the hands and feet < 1.5-mm thick have a low incidence of nodal metastases and are treated effectively with wide excision of the primary with a 1-cm margin. Thicker melanomas are associated with a > 50% rate of regional or systemic failure. In the absence of metastatic disease, these individuals should undergo local excision with a 2-cm margin and intraoperative lymphatic mapping followed by lymphadenectomy if the sentinel node is positive.

Perception of breast cancer risk among women in breast center and primary care settings: correlation with age and family history of breast cancer.

BACKGROUND: A great deal of information about breast cancer risk is available to the public. The accuracy of impressions formed from this information is unknown. METHODS: A total of 750 women attending a breast center and 112 women attending a primary care office completed written surveys of their perceptions of average population risk, personal lifetime risk, and personal 10-year risk of getting breast cancer. Data sufficient to apply the Gail model were obtained, and a calculated estimate of risk was generated. Ratios of perceived to calculated risk were correlated with the respondent's age, family history of breast cancer, and location in a breast center or primary care office. RESULTS: Women in both practice settings overestimated population risk by more than twofold. Eighty percent overestimated personal lifetime risk by more than 50% and 35% by more than fivefold. Only 7% significantly underestimated risk. Ten-year risk estimates were even more inaccurate, with 69% overestimating risk by more than fivefold, 46% by more than 10-fold, and 17% by more than 20-fold. Results from a primary care population were nearly identical. Women at the extremes of age were most inaccurate in estimating risk. It was surprising that family history had little impact on perception of personal risk. CONCLUSIONS: Women in both breast center and primary care settings have a fals:ly high perception of both short-term and long-term breast cancer risk. Health care providers should recognize these misconceptions and be aware that many women may benefit from risk counseling.

Attitudes and opinions toward surgical research. A survey of surgical residents and their chairpersons.

OBJECTIVE: To learn more about how research in academic surgery is viewed by surgical residents and their chairpersons. SUMMARY BACKGROUND DATA: There is a general perception that a productive experience in a basic science laboratory is an important prerequisite for a successful career in academic surgery. METHODS: An anonymous mail survey of 189 surgical residents entering the laboratory and their chairpersons (n=81) was done. Questions included how a laboratory was chosen by the resident, the importance of a basic science laboratory experience as a prerequisite to an academic career, and the perceived goal or goals of the laboratory experience. Data were analyzed by chi square analysis. RESULTS: The response rate from each group was excellent (80% response for residents, 90% from chairpersons). Of the residents surveyed, 78% were men and 22% were women; 51% entered the laboratory after 2 years of clinical training and 34% after 3 years; 84% did their research at their home institution and 91% worked in a surgeon's laboratory; 51% were scheduled to be in the laboratory for 1 year, 41% for 2 years, and 7% for 3 years. Two thirds of the residents were salaried by the surgery department. Both residents (70%) and chairpersons (86%) felt that the best surgical journal was Annals of Surgery. Both groups ranked Science as the top basic science journal. Twenty-four percent of the residents felt their peers offered the best advice in choosing a laboratory compared to 0% of the chairpersons (p<0.01); chairpersons felt they themselves or the program director were better advisors (chairpersons, 44%, vs. residents, 27%; p<0.01). Chairpersons believed that the principal investigators' previous success with residents was the major factor in determining in which laboratory to work; the residents placed more value on their interest in the project. Eighty-nine percent of women requested to go into the laboratory versus 66% of men (p<0.05). Half of the chairpersons and residents believed the faculty felt pressure on them to get grants; however, 71% of postgraduate year (PGY) residents who were PGY3 sensed this pressure compared to 44% of the PGY2 residents (p<0.01). Being in debt did not adversely influence the decision of 77% of these residents to do research. The residents felt more so than did their chairpersons that basic science research was necessary to be a successful academic surgeon (p<0.01). CONCLUSIONS: Although there are some differences in opinions between surgical residents and surgical chairpersons about the value and purpose of basic science research, these differences should be embraced and serve to enhance openness and discussion. Overall, surgical residents viewed the research experience away from clinical surgery as a positive one. The main reason for going into the laboratory was because of a genuine interest in the scientific method and the academic mission.

Development and treatment of pulmonary metastases in adult patients with extremity soft tissue sarcoma.

OBJECTIVE: The authors reviewed a series of adult patients with extremity soft tissue sarcoma to determine the incidence of pulmonary metastases and outcome after treatment. METHODS: Of 716 patients admitted between January 1983 and December 1990, 135 (19%) had isolated pulmonary metastases as the initial site of distant recurrence. Fifty-eight percent (78 of 135) of the patients were treated surgically, and 83% of them had their tumors completely resected. RESULTS: The median survival after complete resection was 19 months; incomplete resection, 10 months; and no operation, 8 months (p = 0.005). The 3-year survival rate after complete resection was 23%, compared with a 2% rate (1 of 57) in those treated nonsurgically (p < 0.001). Factors associated with an increased risk of pulmonary metastases included high tumor grade, tumor size greater than 5 cm, lower extremity site, and histologic type (spindle cell, tendosynovial, and extraskeletal osteosarcoma). Factors associated with complete resectability were the histologic types of spindle cell and extraskeletal osteosarcoma. CONCLUSIONS: Complete surgical resection remains the only possibility for cure from pulmonary metastases in soft tissue sarcoma; however, only 11% of the 19% of patients with an extremity sarcoma whose first distant recurrence is in the lung will be alive at 3 years, despite therapy. Complete resection and the development of more effective adjuvant treatments are imperative to improve outcome for this group of patients.

Recurrence patterns and outcome in 1019 patients undergoing axillary or inguinal lymphadenectomy for melanoma.

Patterns of recurrence and outcome were determined in 403 patients with melanoma who underwent an axillary or inguinal lymphadenectomy. Recurrences developed at single sites in 291 (72%) patients, with a median survival of 11 months, and at multiple sites in 112 (28%) patients, with a median survival of 3 months. Among patients with single-site recurrence, those with nonvisceral recurrence (n = 190) had a median survival of 18.5 months compared with 6 months in those with visceral recurrence (n = 101). Recurrences were treated surgically in 240 (60%) patients, with a median survival of 15 months, and nonsurgically in 112 patients, with a median survival of 4 months. Median survival after complete resection of single-site recurrence was 19 months compared with 6 months after incomplete resection. Multivariate analysis revealed that outcome was improved by surgical treatment, single-site and nonvisceral recurrence, and primary site in an extremity. These observations support an approach of selective resection in the treatment of recurrences after lymphadenectomy.

Effect of hyperbaric oxygen on murine neutrophil and T-lymphocyte functions.

We investigated the effect of repeated hyperbaric oxygen (HBO) exposure on PMN and T-lymphocyte functions in a murine model. Animals received eight 90-min exposures twice daily to 2.4 ata and 100% or 10% oxygen. Control animals were maintained in room air. On the ninth day spleens and peritoneal cell exudates were harvested. Phagocytosis was measured by flow-cytometric analysis of the ability of PMN to engulf formalin-killed, fluorescence-labeled Staphylococcus aureus. PMN-killing capacity was measured by the ability of PMN to undergo an oxidative burst after stimulation with N-formyl methionyl-leucyl-phenylalanine, phorbol myristate, or opsonized zymosan. T-lymphocyte subpopulations were identified using monoclonal antibodies and two-color flow cytometry after 48 h stimulation with phytohemagglutinin, and lymphocyte proliferation was measured by 3 H-thymidine incorporation. We found PMN phagocytosis and oxidative burst were unchanged after HBO treatment. Lymphocyte proliferation was decreased, and an activated population of CD8+ T cells appeared after mitogen stimulation. We conclude that, although PMN function is not affected by prior HBO, lymphocyte proliferation is decreased.

Postburn suppression of murine lymphocyte and neutrophil functions is not reversed by prostaglandin blockade.

Certain arachidonic acid metabolites, including prostaglandins (PGs) E1 and E2, have been shown to exert marked immunosuppressive effects on T-cell and macrophage functions. Cyclooxygenase blockade with indomethacin or ibuprofen may ameloriate these effects. In the current study we measured lymphocyte proliferation by thymidine incorporation, the presence of T-cell activation antigens with monoclonal antibodies and two-color flow cytometry, and neutrophil (PMN) oxidative burst using a fluorescent marker, in control mice and in burned mice treated with indomethacin for 10 days after injury. One-half of the cell cultures were treated with indomethacin in vitro to ensure its continued presence during stimulation. Separate groups of mice were fed a fish oil-based diet which leads to the production of PGE3 rather than PGE2, versus standard mouse chow, a soy-bean oil-based diet which leads to PGE2 production. Lymphocyte proliferation, expression of T-cell activation antigens, and PMN oxidative burst remained depressed in burned mice treated with indomethacin in vivo (plus in vitro) and in those which received the fish oil-based diet, compared to control. Blockade of PG synthesis after murine burn injury by cyclooxygenase inhibition or alterations in the diet failed to restore T-lymphocyte activation or proliferation or to improve PMN oxidative burst. These data suggest that PGE2 alone does not explain the immunosuppression noted after burn injury.

Temporal analysis of murine lymphocyte subpopulations by monoclonal antibodies and dual-color flow cytometry after burn and nonburn injury.

The immune suppression that frequently accompanies severe injury undoubtedly contributes to subsequent infectious complications. Various lymphocyte subpopulations may be identified by surface antigen expression, and alterations in antigen expression by lymphocytes may reflect host immune competence. Using monoclonal antibodies (Moabs) and dual-color flow cytometry, we studied lymphocyte phenotypic expression in mice after either controlled burn injury or hind-limb amputation, with use of peripheral blood, lymph node, and spleen for cell preparation. Moabs were utilized specific for T cells (Lyt-1), helper/inducer cells (L3T4), suppressor/cytotoxic cells (Lyt-2), B cells (IgG), and activated T cells (Ia or IL-2 receptor). The assay techniques called for small amounts of tissue and avoided gradient procedures that might result in selective loss of some lymphocyte populations. The most consistent changes observed were depressions in percentages of L3T4+ and Lyt-2+ cells in spleens of burned mice, accompanied by depression in Ia+ (possibly activated or proliferating) subsets of L3T4+ and Lyt-2+ cells, and the appearance of increased percentages of non-B, non-T lymphocytes. Changes in lymph node cells were minimal. The major alteration seen in peripheral blood was substantial depression of Ia+ subsets, although burned mice had increased circulating Lyt-2+ cells on several late postburn days. Burned mice, unlike limb-trauma mice, had marked splenic hypertrophy with more than a 300% increase in spleen weight after the 30-day postburn period. Eschar excision/implantation experiments indicated that splenic hypertrophy and splenocyte phenotypic changes are related to the presence of burned tissue, which suggests that burned tissue may partially mediate immune changes that accompany severe burn injury.

The effect of thermal injury on murine neutrophil oxidative metabolism.

The ability of polymorphonuclear leukocytes to kill bacteria and yeast is reflected by cellular chemiluminescence or similarly by the production of H2O2 during oxidative metabolism. With the use of flow cytometry and 2'7' dichlorodihydrofluorescein-diacetate, we determined the direct effect of thermal injury and the indirect effect of burn serum on murine polymorphonuclear leukocyte oxidative metabolism after stimulation on days 1, 5, and 10 after 25% total body surface area burn. Control or burn peritoneal leukocytes and 10% control or burn serum were incubated in vitro with 2'7' dichlorodihydrofluorescein-diacetate for 15 minutes, then stimulated with phorbol 12-myristate 13-acetate. The change in polymorphonuclear leukocyte fluorescence was calculated from fluorescence histograms before and after stimulation. The oxidative metabolism of burn polymorphonuclear leukocytes was clearly depressed on days 5 and 10 after burn injury. Control polymorphonuclear leukocytes in the presence of day 5 burn serum produced decreased levels of H2O2, returning to normal by day 10. In general, bactericidal activity is markedly depressed on days 5 and 10 after thermal injury and may be associated with increased risk of sepsis.

Defective T-cell surface antigen expression after mitogen stimulation. An index of lymphocyte dysfunction after controlled murine injury.

Murine spleen T-cell activation in lectin-stimulated cultures after 25% body surface area burn injury or hind-limb amputation was studied by measuring the temporal expression of cell surface markers using monoclonal antibodies and two-color flow cytometry. Lymphocyte activation has been shown to be accompanied by the appearance of new surface antigens, including Interleukin-2 (IL-2) deceptor (IL-2R) and Ia, and emergence of cells that coexpress helper (Th) and suppressor (Ts) surface markers. IL-2R has been shown to appear early on stimulated cells, before DNA synthesis, whereas Ia appears later. Surface markers (L3T4, Lyt2, Ia, and IL-2R) were analyzed at time 0 and after 24, 48, and 72 hours of mitogen-stimulated culture. The appearance of IL-2R and Ia on Th (L3T4+) and Ts (Lyt-2+) populations was markedly depressed after burn injury, but minimal changes were seen after musculoskeletal injury. In addition, coexpression of L3T4/Lyt2 antigens was markedly reduced in burn-derived cells. Serum from burn-injured animals caused depression of surface antigen expression by stimulated normal cells. Recombinant IL-2, when added to burn-derived cell cultures, did not increase expression of these surface markers during culture, nor did it improve proliferation.

Antibody formation after murine injury.

To determine the effect of burn injury on humoral immunity, we followed the murine primary and secondary antibody responses to sheep erythrocytes (SRBC), a T-cell dependent antigen, and lipopolysaccharide (LPS), a T-cell-independent antigen, after 25% TBSA burn. Splenic B-cell-specific antibody synthesis was measured by a hemolytic plaque assay. Simultaneous measurements of specific and nonspecific immunoglobulins were performed by a hemagglutination assay and radial immunodiffusion, respectively. Numbers of splenic primary anti-SRBC-plaque forming cells (PFC) were generally equal in burn and control groups. Numbers of splenic secondary IgG anti-SRBC and anti-LPS PFCs were significantly increased in the burn group with a peak at 5 and 16 days after secondary immunization. There were no differences in serum anti-SRBC or anti-LPS antibody titers between burn-injured and control mice. Nonspecific serum IgG levels were depressed on Days 5 and 10 following injury but had returned to normal by Day 10. Due to the discrepancy between increased secondary PFCs and normal or decreased serum immunoglobulin levels, we measured clearance of exogenously administered IgG. The half-life of IgG in burn mice was 2.5 days compared to 7.1 days in control (P less than 0.05). In conclusion, the antibody response is enhanced after burn injury in the mouse, but this is not reflected in serum levels of specific antibody or immunoglobulins probably due to increased clearance from the circulation.

High dose intravenous immunoglobulin therapy in burn patients: pharmacokinetics and effects on microbial opsonization and phagocytosis.

Depressed serum immunoglobulin levels following severe burns may lead to subsequent infectious complications following such injuries. In a randomized study we administered multiple doses of Sandoglobulin (500 mg/kg) or albumin intravenously to patients with severe burn injuries and closely monitored serum IgG levels. Patients who received IgG therapy had earlier return of normal serum IgG levels compared to control patients; however, control patients attained normal IgG levels during the second postburn week. Serum half-lives of IgG following infusions were remarkably short (means, 47 hours for infusions within 3 days of injury and 154 hours for infusions in the third postburn week); Sandoglobulin has been reported to have approximately a 21-day half-life in normal individuals. We also measured the opsonic capacity of postburn serum, using fluorescein-labeled microbes and flow cytometry; we identified postburn opsonic defects with certain of the organisms as late as 15 days postinjury, even though serum IgG levels had normalized. These defects were corrected by the in vitro addition of Sandoglobulin to the incubation mixture.

Analysis of murine lymphocyte subpopulations by dual-color flow cytometry: technical considerations and specificities of monoclonal antibodies directed against surface markers.

We performed detailed phenotypic analysis of murine lymphocytes from thymus, spleen, lymph node, and peripheral blood using commercially available monoclonal antibodies, each with specificities for membrane surface markers and dual-color flow cytometry. Erythrocyte lysis techniques were utilized for lymphocyte preparation so that inherent difficulties with gradient techniques would be avoided, such as the potential for loss of abnormally sized cells. These studies demonstrated that the specificities of each monoclonal must be carefully determined; for example, the Lyt-1 monoclonal, frequently utilized to identify helper/inducer T cells, also reacts with suppressor/cytotoxic (Lyt-2+) cells; helper/inducer cells are better studied with a more recently available monoclonal, L3T4. Cells from different tissues may differ greatly not only in the presence of surface markers, but also in the surface density of each marker; this density can be studied and quantitated using appropriate analytic software. We also show that larger and more granular lymphocytes appear to be enriched for surface Ia antigen, indicating that these cells may be activated or regulatory subsets; these large, Ia+ T-cells will be lost from analysis if standard, narrow gate settings are used for analyzing forward and side-scatter characteristics or for cell sorting.