Role of I-region gene products in macrophage induction of an antibody response. II. Restriction at the level of T cell in recognition of I-J-subregion macrophage determinants.

The effect of specific anti-I-J reagents on macrophage-T cell interactions was studied in an in vitro antibody response to burro erythrocytes. Macrophages were prepared from the spleens of F1 hybrid mice whose parental strains differed at the I-J subregion. Two F1 hybrids were used for these experiments, [B10.A(3R) X B10.A(5R)]F1 and [B10.S(9R) X B10.HTT]F1. F1 macrophages responded equally well with F1 T-B cells or with T-B cells of either parental strain. When F1 macrophages were pretreated with anti-I-J serum (without complement) specific for one parental haplotype, they were only able to cooperate with T helper (TH) cells of the unblocked haplotype and with F1 TH cells. Identical results were obtained with (Jb X Jk)F1 and (Js X Jk)F1 mice. The results indicate that TH cells possess genetically restricted receptors for macrophage I-J-subregion gene products and that the interaction between this receptor and the macrophage I-J-subregion determinants is essential for the initiation of a primary in vitro antibody response to an erythrocyte antigen.

IR gene regulation of the response to trinitrophenyl-polysaccharides. Two independent genes are required for antibody production.

The primary in vitro antibody response to TNP-Ficoll was found to be under H-2-restricted Ir gene control. Strains B10(H-2b), B10.A(H-2a), and B10.S(9R) (H-2t4) were consistently low responders while strains D2.GD(H-2g2), B10.GD(H-2g2), and B10.S(H-2s) were high responders. The in vitro TNP-Ficoll response in congenic recombinant and F1 hybrid mice demonstrated the requirement for complementation of two independent Ir genes. One Ir gene mapped in or to the left of the I-A subregion with high responder alleles being s or d. The second Ir gene mapped to the right of the I-E subregion and required b or s alleles for complementation. These results were further supported by the ability to block the TNP-Ficoll response by appropriate anti-Ia serum pretreatment of the antigen-presenting macrophages. When a structurally different polysaccharide antigen TNP-dextran was used, an identical pattern of restriction was observed.

Effects of anti-Ia sera on mitogenic responses. III. Mapping the genes controlling the expression of Ia determinants on concanavalin A-reactive cells to the I-J subregion of the H-2 gene complex.

We have shown that the Ia determinants expressed on nylon wool-purified T lymphocytes reactive to concanavalin A (Con A) in serum-free media are coded in a single I subregion of the H-2 gene complex. This region, I-J, is defined by two pairs of intra-H-2 recombinant haplotypes: H-2t3, H-2t4 and H-2i3, H-2i5, carried by B10.HTT, B10.S(9R), B10.A(3R), AND B10.A(5R), respectively. No activity against Con A-reactive T cells has been detected in any antiserum that was produced in strain combinations which shared a common I-J region. This suggests that Ia antigens expressed on Con A-reactive T cells are restricted to the I-J subregion.

Effects of anti-Ia sera on mitogenic responses. II. Differential expression of the Ia marker on phytohemagglutinin and concanavalin A-reactive T cells.

Genes mapping in the I region of the H-2 complex control a system of lymphocyte alloantigens (Ia) which are expressed on a subpopulation of T cells and on most B cells. Specific anti-Ia serum in the presence of rabbit complement removed the splenic T-cell subpopulation responsive to Con-A, but did not affect the response to phytohemagglutinin (PHA) or Leucoagglutinin. Antibodies specific for Ia, H-2K, or H-2D membrane antigens were used without complement to pretreat spleen cells. These antibody pretreated cells responded normally to Con-A and PHA.

Inhibition of immune responses in vitro by specific antiserums to Ia antigens.

Mouse antiserums prepared against Ia antigens, which are products of I (immune response) region genes of the H-2 complex, can inhibit both primary (immunoglobulin M) and secondary (immunoglobulin G) immune responses in vitro by mouse spleen cultures to heterologous erythrocytes. Antiserums directed specifically at products of either the H-2K or H-2D loci have no effect on this response.

Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells.

Several pathways of transmembrane signaling in lymphocytes involve protein-tyrosine phosphorylation. With the exception of p56lck, a tyrosine kinase specific to T lymphoid cells that associates with the T cell transmembrane proteins CD4 and CD8, the kinases that function in these pathways are unknown. A murine lymphocyte complementary DNA that represents a new member of the src family has now been isolated and characterized. This complementary DNA, termed blk (for B lymphoid kinase), specifies a polypeptide of 55 kilodaltons that is related to, but distinct from, previously identified retroviral or cellular tyrosine kinases. The protein encoded by blk exhibits tyrosine kinase activity when expressed in bacterial cells. In the mouse and among cell lines, blk is specifically expressed in the B cell lineage. The tyrosine kinase encoded by blk may function in a signal transduction pathway that is restricted to B lymphoid cells.

Molecular cloning and chromosomal localization of the human homologue of a B-lymphocyte specific protein tyrosine kinase (blk).

A cDNA encoding the human homologue of the murine protein tyrosine kinase, blk, has been cloned from a human B-lymphocyte cDNA library by cross-species hybridization using the murine blk cDNA as a probe. The sequence of the 2608 bp human blk cDNA clone contains an open reading frame encoding a predicted 505 amino acid protein with SH3, SH2 and catalytic domains that contain consensus sequences of the src protein tyrosine kinase family. Comparison of human and murine blk sequences indicated that they share 86% amino acid identity, the most conserved region being the catalytic domain (93% identity). Like the murine blk gene human blk is expressed only in B lymphocytes. The human blk gene was mapped to chromosome 8 at p22-23.

Regional chemotherapy devices: effect of experience and anatomy on complications.

PURPOSE: Regional hepatic arterial infusion (HAI) devices have been used for 17 years, but reports of unacceptably high complication rates have led to controversy about their use. Inadequate or misdirected infusion has been reported to occur in up to 45% of patients. We evaluated whether surgeon experience or presence of variant arterial anatomy related to risk of coagulation. MATERIALS AND METHODS: We reviewed 70 patients undergoing placement of HAI catheters. Surgeons were classed as experienced after 10 procedures and arterial anatomy was evaluated angiographically with confirmation at operation. Complications were categorized as technical (17%) or chemotherapy-related (16%). RESULTS: Inexperienced surgeons had a technical complication rate of 37% (80% of the patients involved had standard anatomy), while experienced surgeons had a technical complication rate of 7% (P < .01). Experienced surgeons had no complications in patients with standard anatomy, while inexperienced surgeons had a 42% (eight of 19) complication rate in similar patients (P < .01). CONCLUSION: We conclude that technical complications are closely associated with surgeon experience and arterial anatomy.

Commentary concerning demonstration of safety and efficacy of investigational anticancer agents in clinical trials.

Expeditious clinical development and approval of new drugs that are beneficial to patients are matters of high priority. There has been a great deal of discussion within the oncology community about what should constitute evidence of effectiveness of new anticancer agents for purposes of drug approval. This commentary is intended to illustrate a variety of end points that can lead to approval of new anticancer agents for specific clinical situations. Although the ultimate hope of antineoplastic therapy is prolongation of life, there are other effects of anticancer drugs that constitute clear clinical benefit and represent evidence of effectiveness. The guiding principle is that the beneficial effects obtained from a new drug should sufficiently outweigh the adverse effects such that the potential risk:benefit ratio achieved by an individual patient is favorable. The assessment of a new drug should flexibly evaluate safety and efficacy in the context of the specific clinical condition being treated. Early discussions with the Food and Drug Administration (FDA) and the National Cancer Institute (NCI) are recommended to identify prospectively the end points and trial designs needed to demonstrate effectiveness of a new drug. The general principles discussed will likely apply to the drug approval process for other medical disciplines as well.

Constant intraperitoneal 5-fluorouracil infusion through a totally implanted system.

Five-day continuous intraperitoneal (ip) infusions of 5-fluorouracil (FU) were injected into five patients as part of a phase I clinical pharmacology study. They received 20 courses through a totally implanted catheter/injection port system. Six courses are evaluable for kinetic parameters and all courses are evaluable for toxicity. In each course a 2 to 3 log FU concentration differential in favor of the peritoneal cavity was achieved and maintained. Steady-state venous plasma FU concentrations averaged 0.34 microM, whereas steady-state ip FU concentrations averaged 697 microM. Mean total body clearance (TBC) in these patients was 18.4 l/min and mean permeability-area (PA) product for diffusion from the peritoneum was 13.7 ml/min. Mean TBC of 20 l/min with ip FU infusion was observed in one patient who also received a 24-hr IV FU infusion for comparison. The TBC during the later infusion was 5.9 l/min. In this patient, calculations indicate 75% extraction of drug during the passage from the peritoneum to the systemic circulation, presumably representing in large part hepatic extraction of FU taken into the portal venous circulation. Ip constant infusion and bolus kinetics were compared in one patient. TBC for the ip bolus was 14.3 l/min, which was approximately half of the TBC of 29.5 l/min determined during the 5-day constant ip infusion. Thus constant ip infusion of FU (l gm/day) can provide an improved regional advantage over bolus ip FU because of an increased TBC. Toxicity was acceptable in all courses. Dose limiting toxicity was regional, namely moderate chemical peritonitis seen in two of the five patients on repeated courses. There was no myelosuppression, alopecia, nausea, or vomiting.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and characterization of a monoclonal antibody against Schistosoma japonicum glutathione S-transferase.

Schistosoma japonicum glutathione S-transferase (GST), expressed from a pGEX plasmid, was isolated from Escherichia coli cells and used to immunize mice in order to generate specific anti-GST monoclonal antibodies. Using a modified immunization and fusion procedure, one stable hybridoma clone secreting an anti-GST antibody (alpha GST-1) was obtained. Milligram quantities of this antibody were produced in vitro in a miniPERM bioreactor and subsequently purified by protein G affinity chromatography. The characteristics of this antibody were investigated by enzyme-linked immunosorbent assays and immunoblotting experiments. The alpha GST-1 antibody was found to react specifically with GST and GST fusion proteins and demonstrated no reactivity with normal E. coli proteins. This monoclonal antibody should be a valuable reagent for tracing the production of GST fusion proteins and possibly for affinity purification of GST fusion proteins.

Chemo-radiotherapy for localized pancreatic cancer: increased dose intensity and reduced acute toxicity with concomitant radiotherapy and protracted venous infusion 5-fluorouracil.

PURPOSE: Although concomitant radiation therapy (RT) and bolus 5-Fluorouracil (5-FU) have been shown to improve survival in locally confined pancreatic cancer, most patients will eventually succumb to their disease. Since 1994, we have attempted to improve efficacy by administering 5-FU as a protracted venous infusion (PVI). This study compares treatment intensity and acute toxicity of consecutive protocols of concurrent RT and 5-FU by bolus injection or PVI. METHODS AND MATERIALS: Since 1986, 74 patients with resected or locally advanced pancreatic cancer were treated with continuous course RT and concurrent 5-FU by bolus injection (n = 44) or PVI throughout the course of RT (n = 30). Dose intensity was assessed for both 5-FU and radiotherapy. Toxicity endpoints which could be reliably and objectively quantified (e.g., neutropenia, weight loss, treatment interruption) were evaluated. RESULTS: Cumulative 5-FU dose (mean = 7.2 vs. 2.5 gm/m2, p < 0.001) and weekly 5-FU dose (mean = 1.3 vs. 0.5 gm/m2/wk, p < 0.001) were significantly higher for patients receiving PVI 5-FU. Following pancreaticoduodenectomy, 95% of PVI patients maintained a RT dose intensity of > or = 900 cGy/wk, compared with 63% of those receiving bolus 5-FU (p = 0.02). No difference was seen for patients with locally advanced disease (72% vs. 76%, p = n.s.). Grade II-III neutropenia was less common for patients treated with PVI (13% vs. 34%, p = 0.05). Grade II-III thrombocytopenia was uncommon (< or = 3%) in both treatment groups. Mean percent weight loss (3.8% vs. 4.1%, p = n.s.) and weight loss > or = 5% of pre-treatment weight (21% vs. 31%, p = n.s.) were similar for PVI and bolus treatment groups, respectively. Treatment interruptions for hematologic, gastrointestinal or other acute toxicities were less common for patients receiving PVI 5-FU (10% vs. 25%, p = 0.11). CONCLUSION: Concurrent RT and 5-FU by PVI was well tolerated and permitted greater chemotherapy and radiotherapy dose intensity with reduced hematologic toxicity and fewer treatment interruptions compared with RT and bolus 5-FU. Longer follow-up will be needed to assess late effects and the impact on overall survival.

Separation of the immune response genes for LDH-B and MOPC-173. III. Evidence that the failure of B10.BASR1 to respond to LDH-B is due to an antigen-presenting cell defect.

Analysis of the immune response of a panel of intra-I-region recombinant mouse strains to LDH-B and MOPC-173 demonstrated that B10.ASR7 (H-2as3) and B10.BASR1 (H-2as4) failed to mount T-cell-proliferative responses to MOPC-173 and LDH-B, respectively. To localize the level of the immune response defect in the B10.BASR1 strain, B10.BASR1 macrophages were shown to be incapable of presenting LDH-B to immune responder B10.ASR7 T cells. These results were confirmed using alloreactivity-depleted and (B10.ASR7 X B10.BASR1)F1 immune T cells. Failure of these strains to respond was shown not to be the result of T cell suppression, because cyclophosphamide and anti-Lyt-2.2-plus-complement treatments did not restore responsiveness. Furthermore, B10.BASR1 macrophages were incapable of educating naive responder T cells in vitro to LDH-B--however, naive nonresponder B10.BASR1 T cells could be educated by responder macrophages to LDH-B in vitro. These results suggest that the failure of B10.BASR1 to respond to LDH-B reflects a defect at the macrophage-T cell interaction level, perhaps related to expression of unique I-A molecules created by intra-I-region recombinatorial events.

In-vitro-derived bone marrow macrophages. Expression of Ia antigens during macrophage differentiation.

Cultures of bone marrow stem cells, grown in the presence of L-cell-conditioned medium, were harvested on successive days to determine the expression of Ia antigens and the acquisition of Ir4 gene-regulated antigen presentation. I-A subregion antigen expression was detected by an indirect radiobinding assay (RBA) as early as day 3 and reached maximal binding at day 7, before declining with additional time in culture. Indirect immunofluorescence (IIF) demonstrated 20% Ia+ cells on day 3 of culture and peaked at 60% on day 7 before declining with continued incubation. A mixed lymphocyte reaction (MLR) across an I region difference was used to assess the kinetics of bone-marrow-derived macrophage ( BMDM ) Ia expression. Maximal stimulation occurred with BMDM stimulator cells obtained from 5-9-day cultures. To investigate the acquisition of Ir gene function, BMDM harvested after various days in culture were used to reconstitute the T cell proliferative response to poly Glu60Ala30Tyr10 (GAT). The ability of BMDM to present GAT was detected after day 5, increased to maximal levels on day 7, and then declined with weak proliferative responses obtained using day 12 BMDM . The presentation of GAT by BMDM was inhibited by monoclonal anti-Ia. 17 antibody. Thus, the acquisition of Ir gene function by BMDM was found to parallel the expression of Ia molecules. Additional experiments were performed to determine whether treatment of BMDM at day 7 with lymphokines or beta-interferon could extend Ia antigen expression. Whereas treatment of day 7 BMDM cultures with Con-A-stimulated rat splenocyte supernatants extended and augmented Ia expression for an additional three days, beta-interferon treatment did not result in augmentation or extension of Ia antigen expression.

Breast feeding and maternal-donor renal allografts. Possibly the original donor-specific transfusion.

Large numbers of maternal lymphocytes are present in breast milk. We asked whether exposure of an infant to maternal lymphocytes during the process of breast feeding would have an effect on the subsequent reactivity of a patient to a maternal-donor related renal transplant. We studied the posttransplant course of 55 patients who had received a primary maternal-donor transplant. Twenty-seven recipients had been breast-fed during infancy and 28 recipients had not been breast-fed. A history of breast feeding was associated with a more favorable posttransplant course as measured by the percentage of patients who had no rejection episodes during the first posttransplant year (P less than or equal to .006). The one-year graft function rate for breast-fed recipients was 82%; this was statistically significantly better than the 57% measured for non-breast-fed recipients (P less than or equal to .05). Statistical significance of differences between groups was not attained when results were evaluated over a five-year interval. A difference between breast-fed and non-breast-fed recipients was not apparent when we evaluated a somewhat smaller group of patients who had received a paternal donor transplant. From these observations we conclude that the process of breast feeding during infancy may result in a measurable immunologic benefit to the recipient of a subsequent maternal-donor related renal transplant.

Perfusion scintigraphy (Tc-99m MAA) during surgery for placement of chemotherapy catheter in hepatic artery: concise communication.

In 17 patients receiving regional hepatic chemotherapy, Tc-99m macroaggregated albumin imaging was used to aid arterial catheter placement and to assess perfusion patterns. Intraoperative imaging with a portable gamma camera allowed immediate monitoring of hepatic and extrahepatic perfusion patterns and assisted catheter manipulation when necessary to achieve optimal flow distribution. In all 12 patients with standard hepatic arterial anatomy, complete perfusion of both lobes of the liver was achieved, although three of them required intraoperative catheter manipulation and repeat imaging after initial placement. The remaining five patients had aberrant hepatic arterial anatomy, and complete perfusion was more difficult to achieve; they exemplified the need for dual catheters, ligation of accessory hepatic branches, and repeated imaging.

Quantitative hepatic arterial perfusion scintigraphy and starch microspheres in cancer chemotherapy.

Hepatic arterial infusion chemotherapy results in a higher concentration of drug delivered to the tumor with less systemic exposure than is possible with intravenous therapy. However, extrahepatic blood flow and/or shunting to the lung can impose a limitation. This study describes a quantitative method for calculating the extrahepatic component and monitoring its changes due to a new adjunctive therapy, degradable starch microspheres (DSM). DSM temporarily occlude the hepatic arterial circulation, thereby increasing the uptake of therapeutic drugs. Twenty patients with metastatic liver cancer underwent hepatic arterial perfusion scintigraphy (HAPS) using Tc-99m MAA to determine blood-flow distribution and to quantitate extrahepatic uptake. The percent shunt index (PSI) was determined at baseline and after each incremental dose of DSM. The baseline PSI ranged from 6-26% (mean 12.3 +/- 5.8 s.d.) and changed progressively after each injection of DSM/Tc-MAA suspension. The patterns of change in shunting are described. Quantitative HAPS provides a means of measuring the extrahepatic component, warns of potential side effects, and helps guide chemotherapeutic decisions.

Definition of hepatic tumor microcirculation by single photon emission computerized tomography (SPECT).

Single photon emission computerized tomography coupled with Tc-99m MAA hepatic-arterial perfusion scintigraphy has been used to examine the density of the functional microcirculation of hepatic tumors relative to normal liver in 24 patients. In both colorectal and carcinoid tumors we have demonstrated an average three-fold greater arteriolar-capillary density in areas of tumor proliferation. The depth of the evoked tumor hypervascularity was found to extend about 4 cm. Tumors greater than 8-9 cm in diameter were uniformly found to have a central hypovascular core. These observations are of importance in the design of selective strategies utilizing therapeutic microspheres directed against the hypervascular proliferating regions of human tumors.

Production of a hybridoma T suppressor cell and suppressor factor.

Two hybridoma T-cell lines were prepared by fusion of burro erythrocyte (BRBC)--specific T suppressor cells with the BW 5147 thymoma line. Culture supernatants from these T cell hybrids specifically suppressed the in vitro IgG antibody response to BRBC. Clones P3E8 and P4B10 were both removed via affinity columns bearing antibodies to BRBC and antibodies to I-Jk subregion determinants. These results indicated that the hybridoma factors were I-Jk determinant bearing and anti-idiotype determinant bearing. P3E8 supernatant was major histocompatibility complex (MHC) restricted, suppressing only H-2k haplotype spleen cells. Clone P4B10 supernatant was not MHC restricted, suppressing k, s, b, and d haplotype spleen cells. The properties of these two hybridoma factors would indicate they are derived from Ts2 cells.

A unique immune response control locus mapping with the H-2D class I region.

The immune response to the polysaccharide antigen Trinitrophenyl (TNP)-ficoll is controlled by two complementing loci in the murine major histocompatibility complex. One locus of control is present in the I-A subregion and the second is located between the S and D regions. A monoclonal antibody 48-21.7 was selected for its ability to significantly block the in vitro primary response to TNP-ficoll. This antibody bound to antigen-presenting cells does not interfere with the presentation of other antigens. Experiments are presented to demonstrate that the monoclonal antibody 48-21.7 precipitates a unique protein of approximately 40,000 daltons. Sequential immunoprecipitation with monoclonal antibodies to the class I Db antigen and to relevant Ia antigens demonstrated no cross-reactivity. Amino acid sequencing has found unique amino acids to be present at three class I N-terminal invariant positions. These observations indicate the presence of a unique class I-like molecule coded for by genes mapping between S and D and involved in the response to the polysaccharide ficoll.