Two common species dominate the species-rich Euglossine bee fauna of an Atlantic Rainforest remnant in Pernambuco, Brazil.

Nowadays, the northern part of the Atlantic Rainforest of Brazil is largely destroyed and forest remnants rarely exceed 100 ha. In a 118 ha forest fragment within a state nature reserve of Pernambuco (Reserva Ecológica Gurjaú), we surveyed the orchid bee fauna (Apidae, Euglossini) using eight different scent baits to attract males. Once a month during one year, the bees were actively collected with entomological nets, from November 2002 to October 2003 by two collectors. We collected 2,908 orchid bee males belonging to 23 species, one of the highest richness values of the Northern Atlantic Rainforest. Bees of only two species, Euglossa carolina (50%) and Eulaema nigrita (25%), which occurred throughout the year, accounted for three quarter of the collected individuals. Both species are typical for open or disturbed areas. Rainforest remnants like those of Gurjaú within the predominant sugar cane monocultures in the coastal plains of the northern Atlantic Rainforest play an important role in orchid bee conservation and maintenance of biodiversity.

Two common species dominate the species-rich Euglossine bee fauna of an Atlantic Rainforest remnant in Pernambuco, Brazil

Abstract Nowadays, the northern part of the Atlantic Rainforest of Brazil is largely destroyed and forest remnants rarely exceed 100 ha. In a 118 ha forest fragment within a state nature reserve of Pernambuco (Reserva Ecológica Gurjaú), we surveyed the orchid bee fauna (Apidae, Euglossini) using eight different scent baits to attract males. Once a month during one year, the bees were actively collected with entomological nets, from November 2002 to October 2003 by two collectors. We collected 2,908 orchid bee males belonging to 23 species, one of the highest richness values of the Northern Atlantic Rainforest. Bees of only two species, Euglossa carolina (50%) and Eulaema nigrita (25%), which occurred throughout the year, accounted for three quarter of the collected individuals. Both species are typical for open or disturbed areas. Rainforest remnants like those of Gurjaú within the predominant sugar cane monocultures in the coastal plains of the northern Atlantic Rainforest play an important role in orchid bee conservation and maintenance of biodiversity.

Resumo Atualmente, a porção norte da Floresta Atlântica brasileira está drasticamente destruída e os remanescentes florestais raramente excedem 100 hectares. Com uso de iscas odoríferas atrativas aos machos, amostramos a fauna de abelhas-das-orquídeas (Hymenoptera: Apidae: Euglossini) em um fragmento florestal de 118 ha na Reserva Ecológica de Gurjaú, Pernambuco. As abelhas foram ativamente coletadas com redes entomológicas por dois coletores, uma vez por mês, de novembro de 2002 a outubro de 2003. Coletamos 2908 machos de Euglossini pertencentes a 23 espécies, um dos mais altos valores de riqueza registrados para a Floresta Atlântica nordestina. Três quartos das abelhas coletadas pertenceram a apenas duas espécies, Euglossa carolina (50%) and Eulaema nigrita (25%), típicas de áreas abertas e perturbadas e ativas ao longo de todo ano. Remanescentes de floresta como os de Gurjaú, em meio às monoculturas de cana-de-açúcar, podem desempenhar um importante papel na conservação das abelhas-das-orquídeas em ambientes fragmentados como os da Mata Atlântica do nordeste do Brasil.

Two common species dominate the species-rich Euglossine bee fauna of an Atlantic Rainforest remnant in Pernambuco, Brazil / Duas espécies comuns dominam a rica fauna de Euglossini em um remanscente de Floresta Atlântica em Pernambuco, Brasil

Abstract Nowadays, the northern part of the Atlantic Rainforest of Brazil is largely destroyed and forest remnants rarely exceed 100 ha. In a 118 ha forest fragment within a state nature reserve of Pernambuco (Reserva Ecológica Gurjaú), we surveyed the orchid bee fauna (Apidae, Euglossini) using eight different scent baits to attract males. Once a month during one year, the bees were actively collected with entomological nets, from November 2002 to October 2003 by two collectors. We collected 2,908 orchid bee males belonging to 23 species, one of the highest richness values of the Northern Atlantic Rainforest. Bees of only two species, Euglossa carolina (50%) and Eulaema nigrita (25%), which occurred throughout the year, accounted for three quarter of the collected individuals. Both species are typical for open or disturbed areas. Rainforest remnants like those of Gurjaú within the predominant sugar cane monocultures in the coastal plains of the northern Atlantic Rainforest play an important role in orchid bee conservation and maintenance of biodiversity.

Resumo Atualmente, a porção norte da Floresta Atlântica brasileira está drasticamente destruída e os remanescentes florestais raramente excedem 100 hectares. Com uso de iscas odoríferas atrativas aos machos, amostramos a fauna de abelhas-das-orquídeas (Hymenoptera: Apidae: Euglossini) em um fragmento florestal de 118 ha na Reserva Ecológica de Gurjaú, Pernambuco. As abelhas foram ativamente coletadas com redes entomológicas por dois coletores, uma vez por mês, de novembro de 2002 a outubro de 2003. Coletamos 2908 machos de Euglossini pertencentes a 23 espécies, um dos mais altos valores de riqueza registrados para a Floresta Atlântica nordestina. Três quartos das abelhas coletadas pertenceram a apenas duas espécies, Euglossa carolina (50%) and Eulaema nigrita (25%), típicas de áreas abertas e perturbadas e ativas ao longo de todo ano. Remanescentes de floresta como os de Gurjaú, em meio às monoculturas de cana-de-açúcar, podem desempenhar um importante papel na conservação das abelhas-das-orquídeas em ambientes fragmentados como os da Mata Atlântica do nordeste do Brasil.

The TNF family member APRIL promotes colorectal tumorigenesis.

The tumor necrosis factor (TNF) family member APRIL (A proliferation inducing ligand) is a disease promoter in B-cell malignancies. APRIL has also been associated with a wide range of solid malignancies, including colorectal cancer (CRC). As evidence for a supportive role of APRIL in solid tumor formation was still lacking, we studied the involvement of APRIL in CRC. We observed that ectopic APRIL expression exacerbates the number and size of adenomas in Apc(Min) mice and in a mouse model for colitis-associated colon carcinogenesis. Furthermore, knockdown of APRIL in primary spheroid cultures of colon cancer cells and both mouse and human CRC cell lines reduced tumor clonogenicity and in vivo outgrowth. Taken together, our data therefore indicate that both tumor-derived APRIL and APRIL produced by non-tumor cells is supportive in colorectal tumorigenesis.

Effect of (-)-Delta(9)-tetrahydrocannabinoid on the hepatic redox state of mice.

(-)-Delta(9)-Tetrahydrocannabinol (Delta(9)-THC), a psychoactive component of marijuana, has been reported to induce oxidative damage in vivo and in vitro. In this study, we administered Delta(9)-THC to healthy C57BL/6J mice aged 15 weeks in order to determine its effect on hepatic redox state. Mice were divided into 3 groups: Delta(9)-THC (N = 10), treated with 10 mg/kg body weight Delta(9)-THC daily; VCtrl (N = 10), treated with vehicle [1:1:18, cremophor EL (polyoxyl 35 castor oil)/ethanol/saline]; Ctrl (N = 10), treated with saline. Animals were injected ip twice a day with 5 mg/kg body weight for 10 days. Lipid peroxidation, protein carbonylation and DNA oxidation were used as biomarkers of oxidative stress. The endogenous antioxidant defenses analyzed were glutathione (GSH) levels as well as enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase (GPx) in liver homogenates. The levels of mRNA of the cannabinoid receptors CB1 and CB2 were also monitored. Treatment with Delta(9)-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (Delta(9)-THC: 8%; VCtrl: 23% increase) and the GSH/oxidized GSH ratio (Delta(9)-THC: 61%; VCtrl: 96% increase), caused by treatment with the vehicle. Delta(9)-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanol/cremophor EL.

Effect of (-)- Δ9 -tetrahydrocannabinoid on the hepatic redox state of mice

(-)-∆9-Tetrahydrocannabinol (∆9-THC), a psychoactive component of marijuana, has been reported to induce oxidative damage in vivo and in vitro. In this study, we administered (∆9-THC to healthy C57BL/6J mice aged 15 weeks in order to determine its effect on hepatic redox state. Mice were divided into 3 groups: (∆9-THC (N = 10), treated with 10 mg/kg body weight (∆9-THC daily; VCtrl (N = 10), treated with vehicle [1:1:18, cremophor EL® (polyoxyl 35 castor oil)/ethanol/saline]; Ctrl (N = 10), treated with saline. Animals were injected ip twice a day with 5 mg/kg body weight for 10 days. Lipid peroxidation, protein carbonylation and DNA oxidation were used as biomarkers of oxidative stress. The endogenous antioxidant defenses analyzed were glutathione (GSH) levels as well as enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase (GPx) in liver homogenates. The levels of mRNA of the cannabinoid receptors CB1 and CB2 were also monitored. Treatment with ∆9-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (∆9-THC: 8 percent; VCtrl: 23 percent increase) and the GSH/oxidized GSH ratio (∆9-THC: 61 percent; VCtrl: 96 percent increase), caused by treatment with the vehicle. ∆9-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanol/cremophor EL®.

Catecholamine synthesis and metabolism in the central nervous system of mice lacking alpha-adrenoceptor subtypes.

BACKGROUND AND PURPOSE: This study investigates the role of alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), on catecholamine synthesis and catabolism in the central nervous system of mice. EXPERIMENTAL APPROACH: Activities of the main catecholamine synthetic and catabolic enzymes were determined in whole brains obtained from alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptor knockout (KO) and C56Bl\7 wild-type (WT) mice. KEY RESULTS: Although no significant differences were found in tyrosine hydroxylase activity and expression, brain tissue levels of 3,4-dihydroxyphenylalanine were threefold higher in alpha(2A)- and alpha(2C)-adrenoceptor KO mice. Brain tissue levels of dopamine and noradrenaline were significantly higher in alpha(2A) and alpha(2C)KOs compared with WT [WT: 2.8 +/- 0.5, 1.1 +/- 0.1; alpha(2A)KO: 6.9 +/- 0.7, 1.9 +/- 0.1; alpha(2B)KO: 2.3 +/- 0.2, 1.0 +/- 0.1; alpha(2C)KO: 4.6 +/- 0.8, 1.5 +/- 0.2 nmol.(g tissue)(-1), for dopamine and noradrenaline respectively]. Aromatic L-amino acid decarboxylase activity was significantly higher in alpha(2A) and alpha(2C)KO [WT: 40 +/- 1; alpha(2A): 77 +/- 2; alpha(2B): 40 +/- 1; alpha(2C): 50 +/- 1, maximum velocity (V(max)) in nmol.(mg protein)(-1).h(-1)], but no significant differences were found in dopamine beta-hydroxylase. Of the catabolic enzymes, catechol-O-methyltransferase enzyme activity was significantly higher in all three alpha(2)KO mice [WT: 2.0 +/- 0.0; alpha(2A): 2.4 +/- 0.1; alpha(2B): 2.2 +/- 0.0; alpha(2C): 2.2 +/- 0.0 nmol.(mg protein)(-1).h(-1)], but no significant differences were found in monoamine oxidase activity between all alpha(2)KOs and WT mice. CONCLUSIONS AND IMPLICATIONS: In mouse brain, deletion of alpha(2A)- or alpha(2C)-adrenoceptors increased cerebral aromatic L-amino acid decarboxylase activity and catecholamine tissue levels. Deletion of any alpha(2)-adrenoceptor subtypes resulted in increased activity of catechol-O-methyltransferase. Higher 3,4-dihydroxyphenylalanine tissue levels in alpha(2A) and alpha(2C)KO mice could be explained by increased 3,4-dihydroxyphenylalanine transport.

Salvage esophagectomy after exclusive chemoradiotherapy: results at the Brazilian National Cancer Institute (INCA).

Surgical resection is considered the gold standard treatment for esophageal cancer, with global cure rates ranging from 15 to 40%. Exclusive chemoradiotherapy has been used for patients with locally advanced esophageal carcinoma or without clinical conditions for esophagectomy, reaching a 5-year survival rate of up to 30%. However, locoregional control is poor, with local recurrence of 40-60%, being reported in the literature. Maybe, these patients can benefit from salvage surgery. In this study, 15 patients with esophageal cancer submitted to salvage esophagectomy after exclusive chemoradiotherapy treatment were retrospectively analyzed. Salvage esophagectomy was demonstrated to be technically feasible. However, it presents with high surgical morbidity. Currently, salvage esophagectomy is considered the best available treatment to attempt cure in cases of tumor recurrence or persistence after exclusive chemoradiotherapy. All the other types of treatments are regarded as palliative with discouraging survival results.

Do consecutive flower visits within a crown diminish fruit set in mass-flowering Hancornia speciosa (Apocynaceae)?

Hancornia speciosa is a self-incompatible, mass-flowering, sphingophilous fruit crop (mangaba) of northeast and central Brazil. The flowers have a precise pollination apparatus, which optimizes pollen transfer between flower and pollinator. While the pollination mechanism avoids self-pollination, mass-flowering promotes geitonogamy. During a flower visit, almost half of the exogenous pollen grains adhering to the proboscis are deposited on the stigma surface. A pollination experiment with a nylon thread simulating six consecutive flower visits within a crown revealed that only the first two flowers visited (positions 1 and 2) are highly likely to set fruit. Super-production of flowers, and consequently obligate low fruit set, seem to be part of the reproductive strategy of the obligate outcrossing plant, Hancornia speciosa.

Effect of genetic modifications by selection for immunological tolerance on fungus infection in mice.

Two strains of mice genetically selected for extreme phenotypes of immunological tolerance to ovalbumin, susceptible (TS) and resistant (TR), were experimentally infected with Sporothrix schenckii. The objective was to observe whether the genetic modifications produced by the selection might be associated with interstrain differences in adaptive immune and innate responses to infection. Therefore, we evaluated the LD(50), CFU, phagocytic index, fungicidal activity, pro-inflammatory cytokines, specific antibody titres, and the delayed-type hypersensitivity reactivity. TR mice were tenfold more susceptible to infection than TS mice, as shown by LD(50) (5 x 10(6) conidia i.v.). In TS mice, the resistance was a consequence of the tissue fungal load reduction, consistent specific T-cell-mediated immunity, and tumour necrosis factor (TNF)-alpha activity at onset of infection. In TR mice, these responses were not precociously detected. Therefore, the absence of CD4(+) T-cell response in the first week of infection might explain the non-clearance of pathogen in TR mice. However, TR mice did show an increase in TNF level and delayed-type hypersensitivity response after the first week post-infection; there was also expansion and increase in granulomatous foci and CFU in the spleen. The expansion of granulomatous foci and the increase in TNF-alpha and tissue fungal load to damaging levels induced severe tissue destruction, general failure of the organs, cachexy and death in TR mice. The results show that genetic selection for extreme phenotypes of immunological tolerance also modified the responses to S. schenckii infection.

Sequestration of inhaled particulate antigens by lung phagocytes. A mechanism for the effective inhibition of pulmonary cell-mediated immunity.

Dendritic cells (DCs) have emerged as the dominant antigen-presenting cells (APCs) of the lung, playing a vital role in the induction of cell-mediated immunity to inhaled antigens. We have previously demonstrated that an airway challenge with the soluble antigen hen egg lysozyme yields rapid acquisition of specific antigen-presenting cell activity by purified pulmonary DCs and a cell-mediated immune response in the lung upon secondary challenge. To examine how a particulate antigen leads to a cell-mediated response in vivo, graded concentrations of heat-killed Listeria (HKL) were injected intratracheally into Lewis rats. The bacteria were rapidly ingested by lung macrophages and polymorphonuclear leukocytes. The ability of purified pulmonary DCs pulsed in vivo by an airway challenge with HKL to subsequently stimulate HKL-specific responses ex vivo showed a threshold response, requiring a dose in excess of 10(9) organisms/rat. By contrast, all dosages of HKL yielded specific sensitization of lymphocytes in the draining bilar nodes. Pulmonary DCs purified from rats after a secondary in vivo airway challenge with HKL at day 14 were ineffective antigen-presenting cells except at high dosages of antigen. The generation of cell-mediated pulmonary inflammation paralleled the antigen-presenting cell activity of pulmonary DCs and was observed only at high antigen dosages. Hen egg lysozyme immobilized onto polystyrene beads and injected intratracheally yielded comparable results to those observed with HKL. We suggest that a pulmonary cellular immune response is generated to an inhaled particulate antigen when the protective phagocytic capacities of the lung are exceeded and antigen is able to interact directly with interstitial DCs. The diversion of particulate antigens by pulmonary phagocytes may help to limit undesirable pulmonary inflammation while allowing the generation of antigen-specific immune lymphocytes in vivo.

The antigen-presenting activities of Ia+ dendritic cells shift dynamically from lung to lymph node after an airway challenge with soluble antigen.

Dendritic cells (DC) are widely distributed in the lung where they are distinguished by their morphology and class II major histocompatibility complex (Ia) antigen expression. Although a role for DC as pulmonary antigen-presenting cell (APC) has been suggested, little is currently known concerning how these cells respond to inhaled antigens in vivo. Hen-egg lysozyme (HEL) was injected intratracheally into Lewis rats; DC were subsequently purified from the lung and regional lymph nodes (LN) at intervals of up to 14 d and examined for their ability to stimulate the proliferation of HEL-immune T cells in vitro in the absence of added HEL. Pulmonary DC displayed APC activities at 3 h and for up to 7 d after the injection of antigen. Dendritic cells in the draining hilar LN showed APC activities that appeared at 24 h, peaked at day 3, and then diminished progressively. After the primary sensitization, HEL-immune T cells were detected in hilar LN but not in the lung. A second airway challenge with HEL at day 14 yielded an antigen-specific pulmonary immune response, characterized histologically by the accumulation of mononuclear cells around lung venules. We conclude that APC activities shift from lung to lymph node during the response to inhaled antigen.

Interferon-gamma and tumor necrosis factor-alpha promote the binding of dendritic cells to fibronectin.

Connective tissue dendritic accessory cells (DC) accumulate at sites of extracellular matrix (ECM) deposition. The adherence of DC to purified ECM proteins was examined. Splenic and pulmonary DC were purified from Lewis rats and incubated on slides coated with fibronectin (FN), laminin (LN), or collagen (COLL) types I, III and IV. In other experiments, DC were pre-incubated with selected proinflammatory cytokines (IL-1, IL-2, IFN-gamma, TNF-alpha) in order to determine their abilities to modulate cell adherence to ECM. Both splenic and pulmonary DC showed dose-dependent binding to FN that was inhibited by the synthetic peptide arg-gly-asp-ser. There was minimal DC binding to LN or COLL. Pretreating DC with IFN-gamma or TNF-alpha enhanced DC binding to FN but did not increase binding to LN or COLL. IL-1 and IL-2 had no demonstrable effect on DC binding to ECM. Our results suggest that FN is a critical ligand for DC in their binding to the ECM. IFN-gamma and TNF-alpha increase adherence of DC to FN and may promote their accumulation in the lung during inflammation.

Blunt and penetrating trauma of the extrahepatic biliary tract

De janeiro de 1973 a dezembro de 1981, 1.483 pacientes foram operados de traumatismo abdominal. Sessenta deles (0,4 por cento) apresentavam trauma das vias biliares extra-hepaticas. A maioria das lesoes eram devidas a feridas penetrantes do abdome (n=50). A vesicula biliar foi o orgao mais frequentemente afetado-51 pacientes (um apresentava avulsao completa da vesicula). O coledoco estava lesado em oito pacientes e um apresentava uma lesao do canal coledoco hepatico comum. Frequentemente, a operacao de emergencia deve ser realizada sem muitos exames pre-operatorios devido a gravidade do traumatismo...

Characterization of the CD4+ and CD8+ tumor infiltrating lymphocytes propagated with bispecific monoclonal antibodies.

To study the CD4+ and CD8+ tumor infiltrating lymphocytes (TIL) in the antitumor response, we propagated these subsets directly from tumor tissues with anti-CD3:anti-CD8 (CD3,8) and anti-CD3:anti-CD4 (CD3,4) bispecific mAb (BSMAB). CD3,8 BSMAB cause selective cytolysis of CD8+ lymphocytes by bridging the CD8 molecules of target lymphocytes to the CD3 molecular complex of cytolytic T lymphocytes with concurrent activation and proliferation of residual CD3+CD4+ T lymphocytes. Similarly, CD3,4 BSMAB cause selective lysis of CD4+ lymphocytes whereas concurrently activating the residual CD3+CD8+ T cells. Small tumor fragments from four malignant melanoma and three renal cell carcinoma patients were cultured in medium containing CD3,8 + IL-2, CD3,4 + IL-2, or IL-2 alone. CD3,8 led to selective propagation of the CD4+ TIL whereas CD3,4 led to selective propagation of the CD8+ TIL from each of the tumors. The phenotypes of the TIL subset cultures were generally stable when assayed over a 1 to 3 months period and after further expansion with anti-CD3 mAb or lectins. Specific 51Cr release of labeled target cells that were bridged to the CD3 molecular complexes of TIL suggested that both CD4+ and CD8+ TIL cultures have the capacity of mediating cytolysis via their Ti/CD3 TCR complexes. In addition, both CD4+ and CD8+ TIL cultures from most patients caused substantial (greater than 20%) lysis of the NK-sensitive K562 cell line. The majority of CD4+ but not CD8+ TIL cultures also produced substantial lysis of the NK-resistant Daudi cell line. Lysis of the autologous tumor by the TIL subsets was assessed in two patients with malignant melanoma. The CD8+ TIL from one tumor demonstrated cytotoxic activity against the autologous tumor but negligible lysis of allogeneic melanoma targets. In conclusion, immunocompetent CD4+ and CD8+ TIL subsets can be isolated and expanded directly from small tumor fragments of malignant melanoma and renal cell carcinoma using BSMAB. The resultant TIL subsets can be further expanded for detailed studies or for adoptive immunotherapy.

Tumour-infiltrating lymphocytes and interleukin-2 in treatment of advanced cancer.

Tumour-infiltrating lymphocytes (TIL) were isolated and expanded from small tumour biopsy samples of twenty-eight patients (thirteen with malignant melanoma, seven with renal cell carcinoma, and eight with non-small-cell lung cancer). The patients were treated with autologous expanded TIL (about 10(10)) and continuous infusions of recombinant human interleukin-2(1-3 x 10(6) U/m2 per 24 h). 29% of the patients with renal cell cancer and 23% of those with melanoma achieved objective tumour responses lasting 3-14 months. Toxic side-effects were limited, and no patient required intensive-care monitoring. Adoptive immunotherapy with TIL and interleukin-2 may be an effective systemic approach to the treatment of some patients with malignant melanoma and renal cell carcinoma.

Direct estimation of the frequency of human cytotoxic T lymphocytes and their precursors following in vitro allosensitization.

Cell mediated lympholysis (CML) has been proposed as an in vitro model of the rejection process that results from transplantation of allogeneic tissue. To date, the absolute frequencies of cytotoxic T lymphocytes (CTL) and their precursors (CTL.P) have not been directly estimated in man because of technical difficulties. Through optimizing the conditions for radiometric detection of 51Cr release and the attendant improvement in CML sensitivity, direct CTL frequency estimates have been determined in peripheral blood (PBL), spleen (SPL), and lymph nodes (LNC) after in vitro allostimulation using unrelated human cells and limiting dilution assays. The mean frequency of CTL generated from PBL is 1 in 826 cells (0.121% +/- 0.101%) which, from preliminary experiments, is significantly greater than that generated from either LNC or SPL (p less than 0.05). With restimulation of primed cells on day 10, the frequency of CTL generated from PBL was increased 400%. The CTL.P frequency (0.0064% +/- 0.0050%) was approximately 5% of the corresponding CTL frequency. The CTL.P frequencies were found to be minimal estimates as both accessory "filler" cells and T cell growth factors increased the level of detection of CTL.P an average of threefold. The limiting cell dilution assay as detailed in this report should be a powerful tool for defining the cellular requirements and related factors necessary for optimal induction of a CTL response and should provide the means for determination of the immunogenetic requirements and the allospecificity of human cytotoxic lymphocytes.