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While tumor-associated antigen (TAA)-specific CD8(+) T lymphocytes have been detected in metastatic melanoma patients, immune response in early disease phases has not yet been carefully evaluated. We looked for circulating cytotoxic T lymphocytes (CTL) directed against Melan-A / MART1, tyrosinase, gp100 and MAGE-3 antigens in patients with a diagnosis of primary cutaneous melanoma by using fluorescent HLA-A2 tetramers. In five out of six cases high numbers of CD8(+)/tetramer(+) cells could be detected by flow cytometry, and in four patients lymphocyte populations specific for two different melanoma antigens (Melan-A/MART1 and tyrosinase) were contemporaneously present. The TAA-specific cells could represent as much as 1/220 T lymphocytes in the circulating CD8(+) population. When tetramers were used to monitor the in vitro expansion of TAA-specific CTL precursors upon antigen-specific stimulation, a diverse expansion potential was evidenced in CTL from the different donors and, more strikingly, in CTL specific for the different TAA. Melan-A/MART1-specific CTL clones derived from two patients exhibited a broad range of avidity. Only the highest avidity clones, representing about 50 % of the cases analyzed, were tumor specific. By correlating tetramer staining with clone avidity, we found that tetramer fluorescence intensity could represent a good indicator of TCR affinity, but not of overall clone avidity.

Background In peripheral blood, myeloid markers identify a heterogeneous mixture of cells in transit from the bone marrow to peripheral tissues. Similarly, HLA-class II DR expression usually identifies mononuclear cells with the potential for developing antigen-presenting activity. We gathered putative antigen presenting cells bearing myeloid markers (My-APC) to study their composition by cell surface phenotype. Methods To gather and dissect My-APC phenotype while excluding lymphocytes and granulocytes, we developed a strategy based on staining red cell-lysed peripheral blood and gating cells bearing myeloid markers and physical parameters of large mononuclear cells. Results Phenotypic analysis within the My-APC gate showed three distinct populations. The largest fraction was constituted by CD14+ monocytes that extended into the other two populations, each expressing gradually lower levels of CD14 surface antigen along with increasing levels of CD16 and CD2, respectively. The CD16 and CD2 expression patterns extended from CD16+CD14+ or CD2+CD14+ double- positive intermediate cells toward each single positive subset, but they were reciprocally exclusive. Interestingly, CD2+CD14- cells within the My-APC gate were equivalent to myeloid dendritic cell precursors (pre-DC) defined previously by the absence of lineage markers and expression of HLA-DR and myeloid markers. Phenotypic analysis of each population revealed differences in the expression of costimulatory molecules and CD62L. Conclusions This novel analytical approach allowed us to distinguish circulating My-APC in three subsets and to identify relationships between monocytes and other related myeloid populations including DC. Cytometry 45:124–132, 2001. © 2001 Wiley-Liss, Inc.

Bone marrow transplantation (BMT) from an HLA-identical donor is an established therapy to cure homozygous β-thalassemia. Approximately 10% of thalassemic patients developed a persistent mixed chimerism (PMC) after BMT characterized by stable coexistence of host and donor cells in all hematopoietic compartments. Interestingly, in the erythrocytic lineage, close to normal levels of hemoglobin can be observed in the absence of complete donor engraftment. In the lymphocytic lineage, the striking feature is the coexistence of immune cells. This implies a state of tolerance or anergy, raising the issue of immunocompetence of the host. To understand the state of the T cells in PMC, repertoire analysis and functional studies were performed on cells from 3 ex-thalassemics. Repertoire analysis showed a profound skewing. This was due to an expansion of some T cells and not to a collapse of the repertoire, because phytohemagglutinin stimulation showed the presence of a complex repertoire. The immunocompetence of the chimeric immune systems was further established by showing responses to alloantigens and recall antigens in vitro. Both host and donor lymphocytes were observed in the cultures. These data suggest that the expanded T cells play a role in specific tolerance while allowing a normal immune status in these patients.

Both tamoxifen and medroxyprogesterone acetate have a direct antitumor effect and are widely used in breast cancer therapy. Luteinizing-hormone-releasing hormone analogs inhibit the growth of breast cancer cells and could represent an alternative treatment for patients affected by breast cancer. Our study was carried out to investigate the effect of leuprorelin (TAP-144) alone or combined with tamoxifen or medroxyprogesterone acetate in human breast cancer cells. Ineffective when used in the absence of estrogens, TAP-144 inhibited the estrogen-stimulated growth of MCF-7, CG-5 and ZR-75-1 cells cultured in medium supplemented with charcoal-treated serum. The growth of estrogen-unresponsive MDA-MB-231 cells was not affected by TAP-144. The combination of TAP-144 with tamoxifen in CG-5 cells did not determine any enhancement of inhibition of cell growth, whereas in both CG-5 and MCF-7 cells, when 1 microM TAP-144 was associated with 0.1 microM medroxyprogesterone acetate, cell growth inhibition was increased, resulting in a subadditive effect. Progesterone receptor levels of CG-5 cells were significantly increased by TAP-144 in the presence of 17 beta-estradiol with respect to those present in control and 17 beta-estradiol-treated cells.

Nonhematopoietic malignant cells may express receptors for hematopoietic growth factors and respond to these peptides. The aim of the present study was to investigate whether small cell lung cancer (SCLC) cells may be stimulated to proliferate by hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), which are currently used in clinical trials in combination with cytotoxic chemotherapy. We studied two SCLC cell lines, H69 and N417. The effects of GM-CSF and IL-3 were evaluated by studying clonal growth, 3H-thymidine incorporation, BUDR/DNA bivariate flow cytometry, c-myc and N-myc oncogene expression, and myeloid surface markers. Our experiments show that both GM-CSF and IL-3 can increase 3H-thymidine incorporation and cloning efficiency and reduce DNA synthesis time of H69 and, to a lesser extent, N417 cells, supporting the hypothesis that hematopoietic growth factors can stimulate the growth of some malignant nonhematopoietic cells in vitro. Further in vitro and in vivo studies are needed to determine whether clinical trials applying these factors for bone marrow recovery after chemotherapy of solid tumors may be hazardous by potentially stimulating growth of remaining tumor tissue.

In the current study we investigated the effect of two different doses of natural interferon-beta (IFN-beta) on steroid hormone receptors in 45 patients with advanced breast cancer. IFN-beta seems to regulate the receptor mechanisms, inducing in cutaneous metastases an increase of oestrogen and progesterone receptors. Moreover, using IFN-beta and tamoxifen as a combined therapy in 23 receptor-positive patients, no negative interference of the two drugs was observed and no relevant side-effects due to the treatment were noticed. The modulation of steroid receptor content by IFN-beta in advanced breast cancer might represent an interesting way to ameliorate the clinical responsiveness to anti-oestrogens.

From February 1987 to January 1989, 60 patients with advanced breast cancer and no prior chemotherapy for advanced disease were randomized and studied, with 31 treated with fluorouracil, epirubicin, and cyclophosphamide (FEC) and 29 patients with fluorouracil, mitoxantrone, and cyclophosphamide (FNC). Doses were 500 mg/m2 fluorouracil, 500 mg/m2 cyclophosphamide, and 50 mg/m2 epirubicin2 or 10 mg/m mitoxantrone, i.v. Day 1 every 3 weeks. There were no statistically significant differences in pretreatment patient characteristics between the groups. Fifty-six patients were evaluable for response (29 in the FEC arm and 27 in the FNC arm). The response rates were 48.2% for the FEC group (complete response (CR) 10.3% and partial response (PR) 37.9%) and 40.7% for the FNC group (CR 3.7% and PR 37%) (not significantly different, NS). The median response duration was 247 and 267 days, respectively (NS), the median time to progression and time to treatment failure was 244 and 155.5 days for the FEC group and 86 and 98 days for the FNC group, respectively (NS). The incidence of nausea/vomiting was 87.1% in the FEC group and 79.3% in the FNC group, with comparable severity. Alopecia occurred in 80.6% of FEC patients and 44.8% of FNC patients (p less than 0.05). The incidences and degrees of severity of leukopenia, anemia, and cardiotoxicity were comparable in the two treatment groups. Efficacy and toxicity of the two regimens were quite similar. FNC can improve the quality of life of patients by providing significantly less alopecia.

Snail medic (Medicago scutellata L.) seeds exhibit a significantly higher content of a trypsin inhibitor than other Medicago species. This inhibitor belongs to the Bowman-Birk family of serine protease inhibitors (BBI) and exhibits a good sequence homology with the BBI from soybean, while presenting some differences. It has been suggested that BBIs have antitumoral and radio-protective activity.In order to assess whether the inhibitor from Medicago scutellata (MsTI) seeds show similar properties to those of BBI from soybean with respect to potentiation of cisplatin-induced cytotoxicity, we evaluated the effects of MsTI on cisplatin-induced cell killing in MCF7 human breast carcinoma cells and HeLa human cervical carcinoma cells.The 24-hour treatment of MsTI in the cell culture medium decreased the clonogenic survival of MCF7 and HeLa cells in a dose-dependent manner and enhanced cisplatin-induced cytotoxicity. The presence of MsTI during the entire incubation period reduced the D37 of cisplatin by 40% in both the cell lines.MsTI could be an useful agent for the potentiation of cisplatin-mediated cancer treatment.

High-dose chemotherapy (HDC) with autologous PBPC-T has been reported to be effective in hematological and in selected solid malignancies. In this setting, infectious complications represent a relevant cause of morbidity.To ascertain the incidence, types and factors influencing the development of early and late infections, we retrospectively analyzed 148 consecutive breast cancer (BC) patients receiving HDC and PBPC-T, both for primary high-risk BC (pBC) and metastatic disease (mBC).Early infection strongly associated with the occurrence of grade 4 mucositis (p0.001), was documented in 28 patients (19%). Late re-activation of varicella zoster virus (VZV) occurred in 14 patients (9%); an inverse correlation between the VZV re-activation and the total amount of T-cells transferred with the graft was observed. Evaluation of immune reconstitution, carried out in 10 out of 148 patients, showed a long-lasting CD+ T-cells depression (2 year), mainly involving the naive CD4+ T-cell subset. Conversely, the analysis of the frequency of proliferating T-lymphocyte precursors, specific for antigens expressed by 3 different widespread pathogens, demonstrated that, notwithstanding the delayed recovery of CD4+ cells, many T-lymphocyte functions were within normal range 1 year after PBPC-T.Altogether these results show that severe mucositis is associated with early bacterial infections and the infusion of large numbers of T-cells plays a role in controlling late VZV reactivation.

The authors describe a case of retrorectal tumour in a 48-year-old female. Retrorectal tumours are rare, and their diagnosis is notoriously difficult and late. In this case the first-step diagnosis was made on the basis of clinical findings and radiological investigations such as abdominal ultrasonography. The second step included pelvic CT scans and MR imaging which yielded more precise anatomical details and information for the best surgical approach. We opted for a posterior approach with complete, easy removal of the tumour mass which, after pathological examination, turned out to be an epidermoid cyst. The postoperative course was uneventful and, one year after operation, the patient complains only of mild, irregular perineal pain with completely negative radiological investigations (US and CT scan).

Recovery of total T cell numbers after in vivo T-cell depletion in humans is accompanied by complex perturbation within the CD8+ subset. We aimed to elucidate the reconstitution of CD8+ T cells by separate analysis of putative naive CD95− CD28+, memory CD95+ CD28+ and CD28− T cell compartments after acute maximal depletion by high-dose chemotherapy (HD-ChT) in women with high-risk breast cancer. We found that recovery of putative naive CD8+ CD95− CD28+ and CD4+ CD95− CD28+ T cells, was compatible with a thymus-dependent regenerative pathway since their recovery was slow and time-dependent, their values were tightly related to each other, and their reconstitution patterns were inversely related to age. By analysing non-naive T cells, a striking diversion between putative memory T cells and CD28− T cells was found. These latter increased early well beyond normal values, thus playing a pivotal role in total T-cell homeostasis, and contributed to reduce the CD4 : CD8 ratio. In contrast, putative memory T cells returned to values not significantly different from those seen in patients at diagnosis, indicating that this compartment may recover after HD-ChT. At 3–5 years after treatment, naive T cells persisted at low levels, with expansion of CD28− T cells, suggesting that such alterations may extend further. These findings indicate that CD28− T cells were responsible for ‘blind’ T-cell homeostasis, but support the notion that memory and naive T cells are regulated separately. Given their distinct dynamics, quantitative evaluation of T-cell pools in patients undergoing chemotherapy should take into account separate analysis of naive, memory and CD28− T cells.

BACKGROUND The immune-mediated graft-versus-tumor (GVT) effect plays a therapeutic role in the treatment of patients with hematologic malignancies who undergo allogeneic hematopoietic stem cell transplantation (HSCT). More recently, it was reported that a GVT effect also occurred in patients who underwent transplantation for metastatic renal carcinoma. The authors carried out a pilot trial of allogeneic transplantation after a reduced-intensity, preparative regimen in patients with refractory malignancies, including solid tumors. The objectives of the current study were to evaluate the feasibility of this approach in terms of toxicity and engraftment and to document evidence of GVT effects. METHODS Seventeen patients with Stage IV malignancies (7 patients with renal cell carcinoma, 3 patients with sarcoma, 2 patients with breast carcinoma, 2 patients with Hodgkin disease, 1 patient with ovarian carcinoma, 1 patient with melanoma, and 1 patient with both melanoma and renal cell carcinoma) that were not amenable to further conventional treatment were enrolled. The median patient age was 43 years (range, 10–60 years). The Eastern Cooperative Oncology Group performance status (PS) was 0–1 in 11 patients and 2–3 in 6 patients. Preparative treatment consisted of reduced-intensity chemotherapy with fludarabine (30 mg/m2 per day for 4 consecutive days) and cyclophosphamide (30 mg/Kg per day for 2 consecutive days) prior to allogeneic HSCT from a human leukocyte antigen-identical sibling. The median number of CD34+ cells infused was 6.06 × 106/kg (range, 1.5–14.0 × 106/kg). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin-A and short-term methotrexate. RESULTS Patients who had a PS of 2–3 prior to undergoing HSCT experienced Grade 4 hematologic toxicities and Grade ≥ 3 organ toxicities and died of either treatment-related complications or disease progression within 100 days from transplantation. By contrast, 10 of 11 patients who had a PS of 0–1 prior to undergoing HSCT experienced only short-lasting, Grade ≤ 3 neutropenia and thrombocytopenia and no organ toxicity; 1 of 10 patients died of graft failure on Day +29 after undergoing HSCT. By Day +90, 100% donor chimerism was documented in all patients with a past history of heavy chemotherapy, whereas mixed donor chimerism was observed in the 4 patients with a past history of only 1 line of chemotherapy and/or immunotherapy prior to entering the HSCT program. Grade 2–3 acute GVHD occurred in 5 patients. Among patients with a follow-up > 100 days, 2 complete responses and 3 transitory partial responses were recorded. CONCLUSIONS With this conditioning regimen, full donor chimerism was achieved rapidly only in patients who had received previous intensive chemotherapy. In a proportion of patients with refractory malignancies, allogeneic transplantation resulted in tumor regression. This novel therapeutic strategy may provide little benefit in patients with poor PS and rapidly progressing disease. Cancer 2002;94:2409–15. © 2002 American Cancer Society. DOI 10.1002/cncr.10491

This study explored the activity of natural interferon-alpha (nIFN-alpha) in regulating cell growth of 6 breast-cancer cell lines. The anti-proliferative effect of the combination nIFN-alpha and tamoxifen (TAM) or medroxyprogesterone acetate (MPA) in CG-5 estrogen-sensitive mammary cancer cells was investigated, and the ability of nIFN-alpha to restore hormone-sensitivity in the MPA-resistant MCF-7 SK sub-line was examined. nIFN-alpha, at concentrations ranging from 10 to 1000 IU/ml, inhibited cell proliferation of all cell lines tested after 3 and 6 days of treatment. In particular, the highest concentration of the drug used was equally effective in hormone-sensitive and in hormone-insensitive cells. A 6-day pretreatment of CG-5 cells with nIFN-alpha, at the above-mentioned doses, sensitized them to the growth-inhibiting activity of subsequent exposure to 10(-7) M TAM or MPA, which resulted in a synergistic effect, and could be explained on the basis of the observed enhancement of estrogen and progesterone receptors due to IFN activity. Conversely, the simultaneous drug combination did not modify the response to the hormone in CG-5 cells. Pre-treatment with nIFN-alpha (from 10 to 1000 IU/ml) restored MPA sensitivity in the MCF-7 SK sub-line, but no modulation of progesterone receptors was seen in this model. The hormone-sensitivity of the parental cell line was not substantially affected by pre-exposure to nIFN-alpha. These data indicate that nIFN-alpha may be potentially useful in enhancing the clinical effectiveness of TAM and MPA and in overcoming hormone resistance.

The significance of steroid receptors (SR) in human brain tumors is presently a field of intense investigation in order to clarify some aspects of the biological behavior of these neoplasms. We studied the relationship between the presence of steroid receptors and the production of metabolites of the arachidonic acid cascade which have been reported to have a role in the biological behavior of some human tumors. We found that some metabolites of arachidonic acid are produced in different amounts in brain tumors which either did or did not express some steroid receptors. In particular the PGE2 were higher in estrogen receptors (ER) positive meningiomas than in ER negative ones and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, is significantly higher in androgen receptors (AR) negative meningiomas than in AR positive ones. In neuroepithelial tumors the glucocorticoid receptors (GR) positive cases synthesized more TxB2 and less PGE2 than the GR negative ones. Our data seem to suggest that some correlations exist between the presence of some steroid receptors and arachidonic acid metabolite production.

Glucocorticoids, mainly dexamethasone (Dex) and methylprednisolone (Mp) are widely used as supportive therapy in the management of intracranial tumors. Their action on cells depends, in part, on the presence of a specific intracellular receptor (glucocorticoid receptor, GR) that binds the hormone leading to a sequence of biological events. In this study, we evaluated the presence of GR and its biological role in neuroepithelial tumors. Tumoral samples from 25 glioblastomas, 18 anaplastic astrocytomas and 14 astrocytomas were analyzed. Glucocorticoid specific binding proteins were detected in 38.6% of the cases using the dextran-coated charcoal method. A correlation was found between grade of anaplasia, sex, and GR positivity. The biological role of this receptor was investigated in 11 primary cell cultures derived from neuroepithelial tumors. For this purpose, different concentrations of Dex and Mp were added to the culture medium. When Dex doses ranging from 2 to 0.016 μg/ml were utilized, a significant stimulation of the cell growth was observed in receptor positive cultures. At the same doses, no modulation of the cell growth was observed in receptor negative cultures. Same responses were obtained when Mp was added to the culture medium.

In the current study, we investigated the effects of natural beta-interferon (beta-IFN) and recombinant alpha-2b-interferon (alpha-IFN) on the growth of the HL-60 cell line. Cells cultured in a medium that contains various concentrations (from 10 to 1000 IU/ml) of interferons showed a growth inhibition, which reaches the maximum after a 6-day treatment, at the highest dose used. Furthermore, we studied the effect of both beta-IFN and alpha-IFN on the level of glucocorticoid receptors. This was enhanced more than 30% with respect to control in HL-60 cells exposed for 24 hours to concentrations of beta-IFN that ranged from 100 to 1000 IU/ml. The increase of the receptor amount was seen even if cells were treated for 5 days, and was not accompanied by a modification of antigen expression of HL-60 cells. alpha-IFN did not modify the glucocorticoid receptor level substantially in our experimental conditions. Our data indicate that both beta-IFN and alpha-IFN regulate HL-60 cell proliferation. Additional studies are required to clarify if modifications of the receptor level induced by beta-IFN could be related to the modulation of hormone-sensitivity in this model.

Iacopino, Fortunata, Robustelli Della Cuna, Gioacchino and Sica, Gigliola, Natural interferon-alpha activity in hormone-sensitive, hormone-resistant and autonomous human breast-cancer cell lines. Int. J. Cancer,71, 1103–1108 (1997) The following grant information was published incorrectly in the article by Iacopino et al. Here is the correct grant information. ACKNOWLEDGEMENTS This work was partly supported by grant 95.00444.PF39 from the “Consiglio Nazionale delle Ricerche”, 1995 and a grant from MURST (60%, 1993). The Authors regret the error.

The effectiveness of an 8-week, 125 mg/day intravenous course of methylprednisolone sodium succinate (MPSS) for improving quality of life in patients with preterminal cancer was investigated in a double-blind, placebo-controlled, multicenter study. Quality of life was assessed using the Nurses' Observational Scale for Inpatient Evaluation (NOSIE), the Linear Analog Self-Assessment Scale (LASA), and the Physicians' Global Evaluation. A total of 403 patients were enrolled: 207 were treated with MPSS and 196 were treated with placebo. MPSS was significantly more effective than placebo in improving quality of life as judged by the changes from baseline in the NOSIE and LASA total scores. (P < 0.05) and by the Physicians' Global Evaluation (P < 0.001). The mortality rate was similar between MPSS-treated males (40.2%), placebo-treated males (35.5%), and MPSS-treated females (40.0%). However, the mortality rate of 27.7% for female placebo-treated females was significantly lower than for their MPSS-treated counterparts. The reason for lower mortality among placebo-treated females is unknown and warrants further study.

The results of controlled clinical trial that used high doses of medroxyprogesterone acetate (MPA) in the treatment of metastatic breast cancer are reported. Two treatment reigmens were used: regimen A, 500 mg daily with a total dose of 30 g; regimen B, 1,000 mg daily with a total dose of 60 g. The overall response rates were similar, with no statistically significant difference between the two treated groups. Regimen A (lower dosage group) reached a remission rate of 44%, whereas regimen B (higher dosage group) had a remission rate of 41%. The mean duration of response was 8 months with regimen A and 9 months with regimen B. The advantages of the lower dosage regimen as opposed to the higher dosage regimen of MPA in the treatment of advanced breast cancer are discussed.A controlled clinical trial that used high doses of medroxyprogesterone acetate (MPA) in the treatment of metastatic breast cancer was conducted. Therapy consisted of 2 treatments: regimen A, 500 mg daily with a total dose of 30 g; regimen B, 1000 mg daily with a total dose of 60 g. From June 1975 to September 1976, 101 patients entered into the study and were randomly allocated into the 2 treatment groups. Both treatment groups were comparable in terms of age, menopausal status, free interval, and dominant site of lesions. Selection of patients was done according to the following criteria: histologically proved progressive metastatic carcinoma of the breast, without any treatment for at least 2 months; no prior hormonal manipulation; performance status 50 or more, and life expectancy longer than 3 months; measurable disease. Overall response rates were similar, with no statistically significant difference between the 2 treated groups. Regimen A reached a remission rate of 44%; regimen B had a remission rate of 41%. The mean duration of response was 8 months with regimen A and 9 months with regimen B. Both regimens were well tolerated. Clinical toxicity was mild with both dosages of MPA. The main side effect was gluteus abscess, with a higher incidence in group B. This was probably due to the greater amount of injected drug suspension in the 1000 mg/day regimen. The incidence of thrombophlebitis and vaginal bleeding was negligible.

Vitiligo patients possess high frequencies of circulating CD8+ T lymphocytes specific for the melanocyte differentiation antigen Melan-A/MART-1. These self-specific T cells exhibit intact functional properties and their T cell receptors are selected for a narrow range of high affinities of antigen recognition, suggesting their important role in the pathogenesis of vitiligo. In order to understand the molecular base for this unexpected, optimal T cell receptor recognition of a self-antigen, a tetramer-guided ex vivo analysis of the T cell receptor repertoire specific for the Melan-A antigen in a patient affected by vitiligo is reported. All T cell receptors sequenced corresponded to different clonotypes, excluding extensive clonal expansions and revealing a large repertoire of circulating Melan-A-specific T lymphocytes. A certain degree of T cell receptor structural conservation was noticed, however, as a single AV segment contributed to the alpha chain rearrangement in 100% of clones and a conserved amino acid sequence was found in the beta chain complementarity determining region 3 of various high affinity cells. We suggest that the conserved alpha chain confers self-antigen recognition, necessary for intrathymic selection and peripheral homeostasis, to many synonymous T cell receptors, whereas the beta chain fine tunes the T cell receptor affinity of the specific cells. In addition, we demonstrate that many high avidity T cell clones from this patient were capable of specifically lysing normal, HLA-matched melanocytes. These autoreactive clones persisted for more than 3 y in the patient's peripheral blood. These data, together with the skin-homing potential of the clones, directly point to the in vivo pathogenic role of melanocyte-specific cytotoxic T lymphocytes in vitiligo.

Accumulation of 99m Tc-Sn-pyrophosphate in pleural effusions has been evaluated in 56 patients grouped as follows: 8 with bacterial effusion (Group A), 27 with malignant effusion treated by local and/or parenteral antitumor chemotherapy (Group B), 21 with malignant effusion treated only by supportive therapy (Group C). Results, expressed as effusion to plasma PPi ratio, ranged from 0.1 to 0.28 in group A, from 0.04 to 0.64 in group B and from 0.60 to 1.73 in group C, with significant differences among the three groups. In no case was uptake found in cells of the sediment. Chemical analysis (including total and ionized calcium, total protein, acid and alkaline phosphatase) of plasma and exudate in neoplastic patients showed a slight, but significant, difference between groups B and C as regards plasma-effusion gradient for total calcium and acid phosphatase. Negative correlation also exists between effusion to plasma PPi ratio and plasma-exudate gradient for ionized calcium in neoplastic patients. The data support the hypothesis that acid phosphatase content and calcium gradient are among the factors involved in the mechanism of PPi accumulation in pleural effusions.

A Phase II clinical evaluation of 4′-epi-doxorubicin has been carried out in 100 patients with various types of solid tumors. Hematopoietic toxicity was dose-limiting but reversible and of mild to moderate degree. Other acute toxic manifestations such as vomiting and alopecia were qualitatively similar to those usually reported for doxorubicin, but lower in frequency and less severe. A number of responding patients received cumulative doses of 4′-epi-doxorubicin in excess of 500 mg/m2. One patient manifested reversible clinical congestive heart failure at cumulative dose of 1,080 mg/m2. Therapeutic activity has been observed in breast carcinoma, in rectal carcinoma and in melanoma. In chemoresistant tumors as rectal cancer and melanoma 4′-epi-doxorubicin deserves further study.

TCR-α and -β chains are composed of somatically rearranged V, D, and J germline-encoded gene segments that confer Ag specificity. Recent crystallographic analyses revealed that TCR-α has more contacts with peptide than TCR-β, suggesting the possibility that peptide recognition predominantly relies on TCR-α. T cells specific for the self Ag Melan-A/MART-1 possess an exceptionally high precursor frequency in human histocompatibility leukocyte Ag-A2 individuals. This provided a unique situation for assessment of the structural relationship between TCR and peptide/MHC ligand at both the pre- and postimmune levels. Molecular and phenotypic analysis of many different Melan-A-specific T cell populations revealed that a structural constraint is imposed on the TCR for engagement with Melan-A peptides presented by HLA-A2, namely the highly preferential use of a particular TCRAV segment, AV2. Examination of CD8 single-positive thymocytes indicated that this preferential use in forming the Melan-A-specific TCR is mainly imposed by intrathymic positive selection. Our data demonstrate a dominant function of TCRAV2 segment in forming the TCR repertoire specific for the human self Ag Melan-A/MART-1 and support the view that Ag recognition is mediated predominantly by TCR-α.

Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells. When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil, stem cell factor, and interleukin-3, 88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost, but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells. After drug exposure, most of the progenitors displayed a CD34+, CD38−, multidrug-resistance (MDR+), Rhodamine 123 low, Hoechst 33342 low phenotype, and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice. In these animals, the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line. These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts.