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To prospectively evaluate the ability of macromolecular contrast medium (MMCM)-enhanced dynamic magnetic resonance (MR) imaging to depict vascular changes in response to cyclooxygenase-2 (COX-2) inhibition of angiogenesis in a human breast cancer model.The institutional committee for animal research approved this study. A human breast cancer cell line, MDA-MB-231, was implanted in 30 female homozygotous athymic rats that were alternately assigned to either a drug treatment group that received celecoxib on a daily basis for 7 days or a control group that received saline. Each animal underwent MR imaging after intravenous administration of a high-molecular-weight contrast agent at baseline and again 24 hours and 7 days after administration. Eleven rats in each group successfully underwent all three studies and had data sets of sufficient technical quality. A bidirectional two-compartment tissue model was used to estimate transendothelial permeability (K(PS)) and fractional plasma volume (fPV) for each tumor. Microvessel density was also measured to enable histologic assessment of angiogenesis. Repeated-measures analysis of variance and unpaired two-tailed t tests were used to evaluate differences in mean values between MR examinations performed in the same rats and between baseline values in treated and control rats, respectively.MR imaging-assayed microvascular K(PS) decreased significantly after 7 days of treatment with celecoxib (P.05), but it was not significantly changed after 7 days in the control group. Likewise, microvascular density, a histologic surrogate of angiogenesis, was significantly (P.05) lower in the treatment group than in the control group. The fPV did not significantly change in either group.Dynamic MR imaging revealed microvascular permeability to a high-molecular-weight contrast agent was significantly reduced by treatment with celecoxib.

Motivation: Although numerous methods have been developed to better capture biological information from microarray data, commonly used single gene-based methods neglect interactions among genes and leave room for other novel approaches. For example, most classification and regression methods for microarray data are based on the whole set of genes and have not made use of pathway information. Pathway-based analysis in microarray studies may lead to more informative and relevant knowledge for biological researchers. Results: In this paper, we describe a pathway-based classification and regression method using Random Forests to analyze gene expression data. The proposed methods allow researchers to rank important pathways from externally available databases, discover important genes, find pathway-based outlying cases and make full use of a continuous outcome variable in the regression setting. We also compared Random Forests with other machine learning methods using several datasets and found that Random Forests classification error rates were either the lowest or the second-lowest. By combining pathway information and novel statistical methods, this procedure represents a promising computational strategy in dissecting pathways and can provide biological insight into the study of microarray data. Availability: Source code written in R is available from Contact: hongyu.zhao@yale.edu Supplementary Information: Supplementary Data are available at

Acute vascular injury that leads to vascular inflammation is a common finding in the preclinical toxicity testing of drugs in rats and dogs. However, the relevance of this finding for risk to humans is unclear. Concern about the safety of these drugs is heightened by the current lack of noninvasive clinical methods to predict the onset of vascular damage in animals or humans. Determining the relevance of this poorly understood preclinical outcome for humans requires a better understanding of the molecular mechanisms of injury in addition to the development of sensitive and specific leading biomarkers for the clinical diagnosis of acute vascular damage. Most molecular research on this toxicity has been performed in rats, but recent development of canine gene expression microarrays makes transcriptomic studies now possible in the dog. In this study, we investigated the molecular mechanisms of drug-induced vascular injury in dogs using gene arrays. After treating Beagles with toxic doses of CI-947, an adenosine receptor agonist, we profiled gene expression in the coronary arteries and correlated those changes with histopathology at 16 and 24 hours after dosing. The results demonstrated that pathobiological processes such as stimulation of the innate immune response, increased extracellular matrix turnover and oxidative stress were active at times of very early injury.

Carboxymethyldextran (CMD)-A2-Gd-DOTA, a slow clearance blood pool contrast agent with a molecular weight of 52.1 kDa, designed to have intravascular residence for more than 1 h, was evaluated for its potential to characterize and differentiate the microvessels of malignant and benign breast tumors. Precontrast single-slice inversion-recovery snapshot FLASH and dynamic contrast-enhanced MRI using an axial T1-weighted three-dimensional spoiled gradient recalled sequence was performed in 30 Sprague-Dawley rats with chemically induced breast tumors. Endothelial transfer coefficient and fractional plasma volume of the breast tumors were estimated from MRI data acquired with CMD-A2-Gd-DOTA enhancement injected at a dose of 0.1 mmol Gd/kg body weight using a two-compartment bidirectional model of the tumor tissue. The correlation between MRI microvessel characteristics and histopathological tumor grade was determined using the Scarff-Bloom-Richardson method. Using CMD-A2-Gd-DOTA, no significant correlations were found between the MR-estimated endothelial transfer coefficient or plasma volumes with histological tumor grade. Analysis of CMD-A2-Gd-DOTA-enhanced MR kinetic data failed to demonstrate feasibility for the differentiation of benign from malignant tumors or for image-based tumor grading.

Objectives: The objectives of this study were to determine if magnetic resonance (MR) estimates of quantitative tissue microvascular characteristics from regions of interest (ROI) limited to the tumor periphery provided abetter correlation with tumor histologic grade than ROI defined for the whole tumor in cross-section. Methods: A metaanalysis was based on 98 quantitative MR image breast tumor characterizations acquired in 3 separate experimental studies using identical methods for tumor induction and contrast enhancement. Results: The endothelial transfer coefficient (K P S ) of albumin (GdDTPA) 3 0 from the tumor periphery correlated (r = 0.784) significantly more strongly (P < 0.001) with the pathologic tumor grade than K P S derived from the whole tumor (r = 0.604). K P S estimates, either from the tumor periphery or from the whole tumor, correlated significantly more strongly with histologic grade (P < 0.01) than MR image estimates of fractional plasma volume (fPV) from either tumor periphery (r = 0.368) or whole tumor (r = 0.323). Conclusions: K P S estimates from the tumor periphery were the best of these measurable MR image microvascular characteristics for predicting the histologic grade.

Successful development and use of biomarkers will improve the productivity of oncology drug development. Recognition of the importance of biomarkers for speeding drug development is reflected in the precise definitions and concepts proposed by an NIH Working Group to standardize terminology and promote a more coherent and systematic approach to the development and use of biomarkers. Potential clinical biomarkers of drug efficacy are often identified through pre-clinical studies or basic research. Identification of potential biomarkers for use in oncology is moving rapidly forward through continuing advances in clinical imaging technologies, especially molecular and functional imaging. Other rapid advances are a product of the growing availability of new scientific reagents for established technologies and of high-throughput genomic and proteomic technologies that can generate hundreds of potential biomarkers for further evaluation. In certain cases, conventional clinical diagnostic techniques or assays can be adapted for use in pre-clinical models to evaluate their ability to serve as biomarkers for predicting clinical responses to new drug candidates. Evaluation (pre-clinical and clinical) of a potential biomarker is often the longest stage of biomarker development, and standards for evaluation or validation depend on the intended use and stage of clinical development. Biomarkers verified for use in preclinical studies can be used to help select appropriate animal models and lead compounds. Biomarkers verified for use in clinical trials can confirm a drug's pharmacological or biological mechanism of action, guide protocol design, aid patient and dose selection, and help to minimize safety risks. Oncology drug development can be optimized by using a tiered set of clinical biomarkers that predict compound efficacy and safety with increasing confidence at each rise in tier thereby aiding corporate decision-making about advancing compounds. In oncology, a special class of extensively evaluated biomarkers of efficacy (surrogate endpoints) that generally correlate with desired clinical outcomes can be used as a basis for corporate decisions as well as for gaining accelerated provisional regulatory approval of a drug.

The aim of this study was to evaluate the potential of dynamic magnetic resonance imaging (MRI) enhanced by macromolecular contrast agents to monitor noninvasively the therapeutic effect of an anti-angiogenesis VEGF receptor kinase inhibitor in an experimental cancer model. MDA-MB-435, a poorly differentiated human breast cancer cell line, was implanted into the mammary fat pad in 20 female homozygous athymic rats. Animals were assigned randomly to a control (n=10) or drug treatment group (n=10). Baseline dynamic MRI was performed on sequential days using albumin-(GdDTPA)30 (6.0 nm diameter) and ultrasmall superparamagnetic iron oxide (USPIO) particles (approximately 30 nm diameter). Subjects were treated either with PTK787/ZK 222584, a VEGF receptor tyrosine kinase inhibitor, or saline given orally twice daily for 1 week followed by repeat MRI examinations serially using each contrast agent. Employing a unidirectional kinetic model comprising the plasma and interstitial water compartments, tumor microvessel characteristics including fractional plasma volume and transendothelial permeability (K(PS)) were estimated for each contrast medium. Tumor growth and the microvascular density, a histologic surrogate of angiogenesis, were also measured. Control tumors significantly increased (P0.05) in size and in microvascular permeability (K(PS)) based on MRI assays using both macromolecular contrast media. In contrast, tumor growth was significantly reduced (P0.05) in rats treated with PTK787/ZK 222584 and K(PS) values declined slightly. Estimated values for the fractional plasma volume did not differ significantly between treatment groups or contrast agents. Microvascular density counts correlated fairly with the tumor growth rate (r=0.64) and were statistically significant higher (P0.05) in the control than in the drug-treated group. MRI measurements of tumor microvascular response, particularly transendothelial permeability (K(PS)), using either of two macromolecular contrast media, were able to detect effects of treatment with a VEGF receptor tyrosine kinase inhibitor on tumor vascular permeability. In a clinical setting such quantitative MRI measurements could be used to monitor tumor anti-angiogenesis therapy.

ILSI-HESI sponsored an international consortium for the evaluation of alternative models, including the Trp53+ /- mouse, for use in short-term carcinogenicity testing of pharmaceuticals. Products of the ILSI evaluation included guidance for protocol design and assay interpretation, spontaneous tumor incidences, diagnostic criteria for common proliferative lesions, and results of assays for pharmaceutical agents that are known human and/or rodent carcinogens and non-carcinogens. Based on the ILSI evaluation, recommended protocol elements for this model include: 26-week study duration, groups ≥15/sex/dose, a positive control group (benzene or p-cresidine), a negative control group and 3 dose groups, the high dose set at MTD or MFD, routine in-life evaluations, and complete necropsies with microscopic evaluation of tissues. Favored statistical analyses are trend tests or pair-wise comparisons, with no adjustments for survival. For an assay to be valid, positive control groups must demonstrate an effect, and the MTD or MFD must be reached in both sexes. Criteria for a negative response include a valid assay, no statistical increase in common tumors, no biologically significant numerical increase in rare tumors, and no tumor incidence above that of historical controls. Positive responses can consist of statistically significant increases in the incidence of a common tumor or numerical increases in a rare tumor, which may not be statistically significant. In either case, the incidence should be clearly above historical control values. Evidence of a dose response or occurrence of hyperplasia in a tissue with a neoplastic response can support interpreting an assay as positive. The two most common spontaneous tumors (>1%) in Trp53+/- mice are malignant thymic lymphomas and subcutaneous sarcomas. Use of implanted electronic transponders can increase the incidence of sarcomas. Important rare spontaneous tumors (incidence ≤1%) are osteosarcomas and pulmonary adenomas. Many other tumor types have been reported to occur sporadically in Trp53+/- mice. Diagnostic challenges for this model include differentiating lymphoma from atypical thymic hyperplasia and recognizing the variable histopathology of subcutaneous sarcomas. In reported bioassays, Trp53+/- mice responded positively to genotoxic carcinogens, negatively to non-genotoxic rodent carcinogens, and negatively to noncarcinogens, indicating that unlike the 2-year mouse assay, this short-term assay is not overly sensitive. Positive responses often elicited an increase in tumors that occur spontaneously. To successfully use this model, pathologists must understand the biology of the Trp53 tumor suppressor gene and the principles of protocol design and data interpretation for short-term bioassays. They must also know the historical response pattern of Trp53+/- mice to test agents and be able to accurately diagnose tumors in this model. Use of the Trp53+/- mouse presents the pharmaceutical industry with several challenges, one of which is managing the uncertainty created by a lack of precedents for regulatory decisions about some possible outcomes for short-term carcinogenicity assays.

The T-cell-specific receptor, CTLA-4, has been demonstrated to be a potent negative regulator of lymphocyte activation, the functional significance of which has been demonstrated in murine tumor models using blocking antibodies. However, the mechanism(s) involved in enhancing tumor regression has not been identified. In this study, we determined whether IFN gamma was playing a role in this activity. In vitro, anti-CTLA-4 enhanced IFN gamma production by lymph node cells obtained from tumor-bearing mice (351 pg/ml vs 77 pg/ml). Additionally, fibrosarcoma-challenged animals treated with anti-CTLA-4 had elevated levels of the IFN-inducible enzyme 2-5-OAS in draining lymph nodes (850 pM vs 260 pM for controls) and an increased amount of IFN gamma in tumor lysates (at day 7, 620 pg/100 micrograms vs 160 pg/100 micrograms in controls). The importance of IFN gamma was demonstrated by the ability of neutralizing antibodies to completely abrogate the anti-tumor effects of anti-CTLA-4. Moreover, fibrosarcoma cells were shown to be exquisitely sensitive to IFN gamma-mediated class I upregulation and histological examination of tumors from anti-CTLA-4-treated mice revealed a trend toward increased tumor cell apoptosis and decreased angiogenesis. These studies have demonstrated that one mechanism for the anti-tumor effects of anti-CTLA-4 relates to its ability to augment IFN gamma production, resulting in an increased expression of class I on the tumor, enhanced apoptosis, and a decrease in blood vessel growth.

The diagnostic potential of ultrasmall superparamagnetic iron oxide particles (USPIO) for quantitative tumor microvessel characterization was assessed by kinetic analysis of dynamic magnetic resonance imaging (MRI) in a rodent breast cancer model. Microvascular characteristics (transendothelial permeability (K(PS)) and fractional plasma volume (fPV)) were estimated in 32 female Sprague Dawley rats, bearing breast tumors of varying malignancy. These values were compared to a prototype macromolecular contrast medium standard, albumin-(GdDTPA)(30). Transendothelial permeability (K(PS)) correlated significantly (P < 0.05) with the tumor grade (Scarff-Bloom-Richardson (SBR) score) for the USPIO (r = 0.36), as well as for the reference macromolecule, albumin-(GdDTPA)(30) (r = 0.54). Estimates for the fPV did not show a statistically significant correlation with the tumor grade for either contrast medium. In conclusion, USPIO-enhanced MRI data were capable to characterize tumor microvessel properties in this breast cancer model: microvascular permeability (determined using USPIO) correlated significantly with tumor grade. Thus, quantitative estimation of microvascular characteristics in tumors could provide a surrogate of new vessel formation (angiogenesis) and thus a further important clinical indication for USPIO, in addition to MR angiography. J. Magn. Reson. Imaging 2001;13:882-888.

A new contrast medium, MS-325, was compared to albumin-(Gd-DTPA)(30) in 18 chemically induced rat breast tumors based on quantitative estimates of microvascular permeability (K(PS)) and fractional plasma volume (fPV) using a two-compartment bidirectional model. No significant correlation was found between MS-325-enhanced microvascular assays with either tumor grade or with microvascular counts (MVCs). In comparison, the correlation coefficient between K(PS) and histologic tumor grade using albumin-(Gd-DTPA)(30) (r =.58) was statistically significant (P

Characteristics of the cell nuclei within human breast tumors, as assayed and scored by the Scarff-Bloom-Richardson (SBR) histopathologic method, correlate with patient prognosis and long-term survival rates (1,2). We have shown previously in rodent mammary tumors that the histopathologic tumor grade correlates with quantitative magnetic resonance imaging (MRI)–derived estimates of tumor microvascular permeability obtained using a prototypic macromolecular contrast medium, albumin–(GdDTPA)30 (3). Microvessel permeability depends on functional and morphologic characteristics of cancer vessels and on physicochemical properties of the injected solute/ contrast medium molecule (4–7). In this study, we focus on specific characteristics of two contrast media and their influence on MRI-derived estimates of microvessel characteristics. The increased permeability of tumor vessels to macromolecular solutes has been demonstrated repeatedly using albumin–(Gd-DTPA)30, which is used in this study as a reference standard (8–10). Although not previously tested as probes of endothelial hyperpermeability, ultrasmall superparamagnetic iron oxide (USPIO) particles, having a mean diameter of approximately 20 nm, were compared in this study to albumin–(Gd-DTPA)30, which is approximately 20% the size of USPIO. Unlike albumin–(Gd-DTPA)30, which is incompletely eliminated and potentially immunogenic, USPIO particles have a favorable pharmacologic and tolerance profile and are now being tested clinically (11). Thus, the purpose of this study was to define the potential of USPIO (represented by NC100150 Injection [Nycomed Imaging AS, Oslo, Norway]) for the quantitative characterization of tumor microvasculature, specifically for measures of the microvessel permeability and the plasma volume. Results were compared to estimates derived with the established macromolecular contrast medium, albumin–(Gd-DTPA)30, and to the histologic tumor grade.

Amplification and overexpression of erbB2 (Her-2/neu) proto-oncogene has been linked to human malignancies including tumors of the breast, ovary, and stomach. It has been implicated in tumor growth, sensitivity to standard chemotherapy, prognosis of patients, and disease-free survival. Although the clinical use of trastuzumab (Herceptin) has prolonged the survival of breast cancer patients with erbB2-overexpressing tumors, there is an urgent need for more potent and orally bioavailable small-molecule inhibitors. CP-724,714 is a potent inhibitor of erbB2 receptor autophosphorylation in intact cells and is currently undergoing phase I clinical trials. Here, we describe the effects of CP-724,714 in vitro and in vivo in human breast cancer models. CP-724,714 is selective for inhibiting growth of HER2-driven cell lines. In addition, we show that it induces G1 cell cycle block in erbB2-overexpressing BT-474 human breast carcinoma cells and inhibits erbB2 autophosphorylation in xenografts when administered p.o. to athymic mice. It induces a marked reduction of extracellular signal–regulated kinase and Akt phosphorylation, tumor cell apoptosis, and release of caspase-3. P.o. administration (q.d. or b.i.d.) of CP-724,714 inhibits the growth of erbB2-overexpressing tumors in athymic mice without overt adverse effects. [Cancer Res 2007;67(20):9887–93]

This chapter deals with the new animal models in toxicology. The field of in vivo genetic engineering is evolving rapidly. The chapter illustrates that some of the gene-altered models can be invalidated for their intended uses in toxicology or simply abandoned for a variety of technical reasons. However, genetic technologies continues to improve to allow the creation of ever more sophisticated genetically altered models, which can replace those rejected for use in toxicology research and testing. Many of the new gene-altered mice, like the mouse models of the first genetically engineered generation, will be unique and valuable tools for the in vivo study of toxicological problems. Perhaps most significantly, they will continue to be useful for the in vivo confirmation of in vitro research findings. The new models can be designed to provide more accurate and relevant information for solving problems in toxicology and to significantly increase the predicitivity of animal testing for determining human toxicological risk.

The diagnostic potential of a new rapid clearance blood pool contrast medium (P792; MW = 6.47 kDa) for the MR assessment of microvessel characteristics was assessed in 42 chemically-induced breast tumors, with comparisons to albumin-(Gd-DTPA). Microvessel characteristics, including the transendothelial permeability (K(PS)) and the fractional blood volume (fPV), were estimated by using dynamic MR data fit to a bidirectional two-compartment model. The MR-derived estimates for K(PS) and fPV using each contrast agent were compared, and assays using each contrast agent were correlated to the histologic tumor grade (SBR score) and the microvascular density (MVD) counts. Using P792-enhanced data, neither K(PS) nor fPV showed a statistically significant correlation with the tumor grade or the MVD (P >.05). Conversely, using albumin-(GdDTPA)(30), K(PS) values correlated significantly with the histologic tumor grade (r =.55; P

Most nonclinical safety studies performed to satisfy regulatory requirements include numerous anatomic and clinical pathology endpoints. Consequently, the traditional predominant role of the pathologist in the pharmaceutical industry has focused upon the interpretation of histologic changes in tissues and of clinicopathologic changes in blood from animals that had been exposed to new chemical entities. In addition to this conventional role, pathologists are increasingly being recognized for their expanded contributions to drug discovery and development. The medical training, specialty or residency training, and comparative medicine perspective place the industrial pathologist in a unique position to contribute broadly to the objectives of drug discovery and development programs. In addition, the technologies in the pathologist’s repertoire have broad application and utility to many areas of biomedical research in the pharmaceutical industry. Such objectives and areas of research include characterization of ! disease states; identification of modified disease states as a response to administration of potential therapies; characterization and quantification of toxic responses to drug administration ; delineation of mechanisms of toxicities and potential relevance to target species; and facilitation of multidisciplinary efforts to monitor for the clinical occurrence, progression, and reversibility of adverse events. The business and science of pharmaceutical research and development is increasingly complex, and the pressures associated with them create competitive advantages for companies that are able to deploy resources in novel and beneficial ways. Use of pathologists to support drug discovery represents one such approach.

Publisher Summary This chapter discusses the form(s) of transfer factor (TF) that are released by viable leukocytes from tuberculin responsive human donors when their leukocytes are incubated with PPD in vitro . Data obtained from studying this antigen-liberated (or released) has provided further insight into the roles of TF–H5, TF–H7, and dephosphorylated TF–H5 in the induction of antigen specific responsiveness. The chapter concludes that when immune lymphocytes are incubated with specific antigen they release only dephosphorylated TF–H5. Dephosphorylated TP–H5 is apparently derived from the cell surface (or secreted after it is derived from TF–H5) and also is apparently released nonspecifically by lymphocytes incubated for prolonged periods at 37°C as part of a normal turnover process of cellular membrane constituents. It is speculated, however, that cell-free supernatants derived from lymphocytes merely incubated at 37°C for 4 h without specific antigen added would contain a family of TFs representing the range of antigen sensitivities found in the donor.

Nonspecific cytotoxic cells (NCC) from the catfish (Ictalurus punctatus) may comprise a population of cells that are responsible for cellular immunity in the fish. NCC kill a wide variety of transformed target cells, and previous studies have indicated that NCC share properties with mammalian natural killer cells. In the present study, many biophysical and biochemical properties of NCC were defined. NCC were nylon wool nonadherent and adherent. NCC activity was also enriched in plastic nonadherent cells. NCC were nonphagocytic (for carbonyl iron), and they did not bind to Sephadex G-10. Characterization of NCC by density gradient centrifugation indicated that they comprise a relatively homogenous population of cytolytic cells that band at 45.5% Percoll. Moderate to high doses (500-2500 R) of X-irradiation produced a stimulatory effect on NCC lysis of labeled target cells. Additional studies indicated that a soluble suppressor protein in catfish serum (CFS) regulated NCC activity. This S. aureus protein A binding component isolated from CFS suppressed NCC activity. Analysis by SDS-PAGE indicated that the soluble regulatory protein had properties similar to immunoglobulin. These data indicate that NCC share some biophysical properties with mammalian natural killer cells. In addition, NCC appear to be under partial cell regulation by a radiation sensitive suppressor cell and also by a soluble regulator serum immunoglobulin component.

Publisher Summary The dialyzable leukocyte extracts (DLE) prepared from burros immunized with either Mycobacterium tuberculosis (PPD–S) or Coccidioides immitis (spherulin) antigen can transfer antigen-specific cell-mediated immunity (CMI) to previously nonresponsive human lymphocytes in vitro. The results of experiments described in this chapter indicate that when Burros are immunized with either Mycobacterium tuberculosis antigen or Coccidioides immitis antigen, DLE subsequently prepared from their PBL contains TF activity, which is active in inducing antigen-specific responsiveness in previously nonimmune human lymphocytes. Burro TF activity easily can be detected using the LMI assay, the in vitro assay for human TF activity. Characterization of Burro TP specific for PPD–S indicates that it has physical–chemical properties similar to those of human TF derived from DLE.

Publisher Summary This chapter presents immunochemical and physical–chemical evidence for the presence of thymosin alpha1-peptide in dialyzable leukocyte extracts (DLE). In a study described in the chapter, the collective results obtained from immunologic and physical–chemical evaluations of the E-receptor regeneration promoting activity present in human DLE indicate that it is partly because of the presence of thymosin α1 peptide in DLE. There are, however, additional components in DLE that express a similar activity but do not seen to be intact thymosin α1. Included here might be thymic humoral factor, which has a pi of 5.7 and could be responsible for the activity found in the β region when DLE Sephadex G-25 Fraction IV was fractionated by IEP in a 2.5 to 10 gradient.The documentation of the presence of a variety of T-lymphocyte maturation or differentiation factors in DLE explains why DLE may have nonspecific beneficial effects when it is employed as an immunotherapeutic agent.

Publisher Summary The leukocyte migration inhibition (LMI) assay can be used to detect and quantitate transfer factor (TF) in dialyzable leukocyte extracts (DLE) in vitro . The development of this in vitro assay for TF has in turn permitted the development of a new method for determining the TF potency in different preparations of DLE. The chapter describes and other clinical applications of the LMI assay: (1) documentation of antigen selective defects in cell-mediated immunity (CME), (2) selection of suitable donors for leukocytes for the preparation of DLE for immunotherapy, (3) prediction of the patient's in vivo response to DLE therapy, (4) determination of optimal dosages of DLE for immunotherapy and immunoprophylaxis, and (5) monitoring the patient's response to treatment with DLE. The chapter presents case studies in which DLE is employed containing antigen-specific TF to treat two patients with antigen-selective defects in CMI to either Mycobacterium fortuitum or Candida albicans infections, respectively.

Cultured mononuclear cells (MNC) from individuals homozygous or heterozygous for the defective gene causing the inherited disease cystic fibrosis (CF) synthesize three unusual "mediators" termed ciliary dyskinesia substances (CDS), which markedly affect tracheal mucociliary systems in vitro. MNC cultures from normal healthy controls do not accumulate any CDS, whereas MNC cultures from non-CF patients controls with pulmonary disease synthesized at least one CDS. The possible involvement of the CDS in pulmonary disease is being investigated. In this study, we sought to determine whether the CDS could be chemoattractants for polymorphonuclear neutrophils (PMN), since they have characteristics in common with known chemoattractants generated by alveolar macrophages. Our analyses of crude MNC culture supernates indicated that cultures from both CF genotypes accumulate significantly higher levels of PMN chemoattractants than do analogous cultures from normal healthy controls. CF homozygote MNC also generated more activity than MNC from patient controls with chronic pulmonary disease. Fractionation of MNC culture supernates by gel permeation chromatography and characterization of active fractions demonstrated six distinct PMN chemoattractants in cultures from CF genotypes; five were also present in patient control and four in normal healthy control cultures. The excessive chemoattractant activity in MNC cultures from CF genotypes and patient controls was due to several different substances produced by monocytes: (a) two components of 1,000-3,500 mol wt. (b) two fragments of C5, and (c) a fragment of C3. One C5 fragment had ciliary dyskinesia activity, the other did not. The C3 fragment chemoattractant also had ciliary dyskinesia activity and was not found in MNC cultures from patient controls. A third CDS, Which is CF-specific (5,000 mol wt), was neither chemotactic not chemokinetic and did not inhibit random PMN migration; however, fractions containing this CF-specific CDS completely inhibited PMN chemotaxis in response to three different chemoattractants. We conclude that all of the CDS can potentially play a role in the pathophysiology of lung disease, as judged by their effects on PMN movement in vitro.