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Infections are a major cause of morbidity and mortality in multiple myeloma (MM), a cancer of the immune system. Vaccination clinical efficacy endpoints have not been demonstrated, and there are limited data on surrogate markers of efficacy. This pilot study evaluated sequential immunologic markers after standard pneumococcal vaccination (PV) in patients with MM and non-MM controls. Vaccination was standard for PV (PCV13 or PPV23), with laboratory testing at baseline and at 2, 4, 12 and 24 weeks after vaccination. Immunoglobulin G (IgG) antibodies to pneumococcal antigens were detected by ELISA. Prevaccination total IgG levels and IgG subclass levels were also measured by ELISA. Four of 6 controls responded with at least a 2-fold increase in antibody concentration; only 2 controls had a sustained increase in concentration. Six of 8 patients with MM had at least a 2-fold antibody increase; however, only 2 of these patients showed a sustained increase of antipneumococcal antibody. Response rate differences were not statistically significant in this small pilot, and there was no relationship between responsiveness to PV and initial serum total IgG levels or IgG subclasses at study entry. Future prospective studies are needed to ascertain the immunological and clinical efficacy and effectiveness of various vaccines and vaccination strategies in MM., Competing Interests: Conflicts of Interest Michael Thompson serves on the myeloma and indolent lymphoma committees of Via Oncology, LLC (Pittsburgh, PA); medical advisory board of AIM Specialty Health (Deerfield, IL); scientific steering committee for the Connect® MDS/AML Registry (Celgene Corporation, Summit, NJ); and multiple myeloma registry of Takeda Oncology (Cambridge, MA).

Purpose: Common reasons for hospitalization and death in patients with multiple myeloma (MM) are infections. As patients with MM are living longer and are treated with immunomodulatory drugs, there is a need to immunize against vaccine-preventable diseases and ultimately determine the efficacy of these vaccines. We evaluated vaccination practice patterns in MM patients at our health system using electronic medical records and data analytics., Methods: This institutional review board-approved study retrospectively reviewed patients with MM who visited the health system from May 2012 to May 2014. Data collected included demographics, influenza vaccination (FV) and pneumonia vaccination (PV) history, hospitalization episodes and associated costs, and duration of survival. Patients were considered PV-positive if vaccinated within 5 years prior to study. FV was defined as optimal (two FV in 2012-2014), suboptimal (one FV in 2012-2014) or none (in 2012-2014)., Results: Of 411 MM patients, 55% were male and 85% Caucasian. Nearly 58% received PV in the past 5 years. FV was 15% optimal, 52% suboptimal and 33% none. A total of 444 hospitalizations involving 204 patients were observed over 2-year follow-up. More than $23 million was incurred from hospitalizations in the 2-year study period. There was no statistically significant difference in all-cause hospitalization and overall survival by FV and PV status., Conclusions: Despite recommendations of vaccination in multiple myeloma, our cohort had low rates of influenza and pneumonia vaccination. FV and PV status did not show any significant association with additional hospitalization or overall survival in this pilot study. Future prospective studies are needed to ascertain the immunological and clinical efficacy and effectiveness of these vaccines in immunosuppressed patients., Competing Interests: Conflicts of Interest Michael Thompson serves/served: as co-chair of the medical oncology multiple myeloma and indolent lymphoma committees of Via Oncology, LLC (Pittsburgh, PA); on the medical advisory boards of AIM Specialty Health (Deerfield, IL) and Doximity, Inc (San Francisco, CA); on the elotuzumab medical advisory board of Bristol-Myers Squibb Co (New York, NY); on scientific steering committees for the Connect® MDS/AML Registry of Celgene Corporation (Summit, NJ); and on the multiple myeloma registry of Millennium Pharmaceuticals, Inc (Cambridge, MA), a subsidiary of Takeda Pharmaceutical Co Ltd.

Multiple myeloma is a cancer of the immune system. Infection is a major cause of morbidity and mortality in patients with multiple myeloma. Some of these infections are preventable by vaccines available to the general population. However, little is known about the clinical effectiveness of these vaccines in patients with multiple myeloma, and the cellular and humoral immune response to vaccination has not been well characterized, especially in conjunction with modern myeloma therapies. The present report reviews the basics of multiple myeloma and the immune system, the available evidence on the immunologic response of patients with multiple myeloma after vaccination, and current practice recommendations regarding specific vaccines. Understanding the immune response to vaccines could help us understand how immuno-oncology-based therapies work in multiple myeloma and provide future directions for research., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population., (Copyright © 2012 International Society for Advancement of Cytometry.)

Background: CTLA-4 was initially described as a membrane-bound molecule that inhibited lymphocyte activation by interacting with B7.1 and B7.2 molecules on antigen presenting cells. Alternative splicing of mRNA encoding the CTLA-4 receptor leads to the production of a molecule (sCTLA-4) that lacks a membrane anchor and is therefore secreted into the extracellular space. Despite studies finding that people with autoimmune disease more frequently express high levels of sCTLA-4 in their blood than apparently healthy people, the significance of these findings is unclear., Methods: Molecules isolated from blood using CTLA-4 specific antibodies were analyzed with ligand binding assays, mass spectroscopy, and biochemical fractionation in an effort to increase our understanding of CTLA-4 immunoreactive material., Results: Mass spectroscopy analysis of the molecules recognized by multiple CTLA-4-specific antibodies failed to identify any CTLA-4 protein. Even though these molecules bind to the CTLA-4 receptors B7.1 and B7.2, they also exhibit properties common to immunoglobulins., Conclusion: We have identified molecules in blood that are recognized by CTLA-4 specific antibodies but also exhibit properties of immunoglobulins. Our data indicates that what has been called sCTLA-4 is not a direct product of the CTLA-4 gene, and that the CTLA-4 protein is not part of this molecule. These results may explain why the relationship of sCTLA-4 to immune system activity has been difficult to elucidate.

Background: Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an important downregulatory molecule expressed on both T and B lymphocytes. Numerous population genetics studies have documented significant associations between autoimmune diseases and single nucleotide polymorphisms (SNPs) within and around the CTLA-4 region of chromosome 2 in man. Furthermore, circulating levels of a soluble form of CTLA-4 (sCTLA-4) have been reported in a variety of autoimmune mediated diseases. Despite these findings, the relationship between levels of sCTLA-4 protein, mRNA transcript levels, and SNPs within the CTLA-4 region have not been clearly defined. In order to further clarify this relationship, we have tested four different SNPs within the CTLA-4 region among subjects whom are negative (n = 53) versus positive (n = 28) for sCTLA-4., Results: Our data do not support a clear association between sCTLA-4 levels and any of the four SNPs tested., Conclusion: The variation in the SNPs tested does not appear to effect sCTLA-4 protein levels, despite reports that they affect sCTLA-4 mRNA.

Cytotoxic T lymphocyte antigen 4 (CTLA4) is a potent inhibitory co-stimulatory molecule believed to be involved in type 1 diabetes and other autoimmune diseases. An association has been reported of both mRNA expression and serum levels of the soluble splice variant of CTLA4 (sCTLA4) with type 1 diabetes. Furthermore, recombinant fusion proteins CTLA4Ig and LEA29Y have been proposed as therapies for type 1 diabetes. We studied the role of (s)CTLA4 in islet autoimmunity. Binding capacity of the proteins to antigen-presenting cells was determined by flow cytometry in competition and binding assays. Functionality of sCTLA4 as well as the therapeutic inhibitory fusion proteins CTLA4Ig and LEA29Y was measured in a dose-response lymphocyte stimulation test, using a panel of diabetes-associated T cell clones reactive to islet autoantigens. As controls, mixed lymphocyte reactions (MLR) were performed to assess functionality of these proteins in a primary alloreactive setting. All three CTLA4 molecules were able to bind to antigen-presenting cells and inhibit the expression of CD80/CD86. sCTLA4 was able to suppress proliferation of different committed autoreactive T cell clones in a dose-dependent manner, whereas CTLA4Ig and LEA29Y were not. Conversely, CTLA4Ig and LEA29Y, rather than sCTLA4, were able to suppress naive alloreactive proliferation in a MLR. Our results indicate a differential role for sCTLA4, CTLA4Ig and LEA29Y proteins in memory versus primary immune responses with implications for efficacy in intervention therapy.

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4-Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4-Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is important for downregulation of T-cell activation, and CTLA-4 gene polymorphisms have been implicated as risk factors for rheumatoid arthritis (RA). Previous studies of the association between the +49 polymorphism of the CTLA-4 gene in RA have provided conflicting results. In order to determine association of the CTLA-4 gene with RA in Chinese Han population, we used denaturing gradient gel electrophoresis (DGGE) to genotype polymorphisms of four SNPs (MH30, +49, CT60 and JO31) of the CTLA-4 gene in 326 RA patients and 250 healthy controls. Furthermore, meta-analysis of all available studies relating +49 polymorphism to the risk of RA was performed to confirm the disease association. Among the SNPs examined, the genotype frequencies of CTLA-4 +49 and CT60 in RA patients differed significantly from controls (P=0.028 and 0.007). In addition, the distribution of four haplotypes constructed by these two SNPs was significantly different between patients and controls (chi(2)=10.58, d.f. =3, P=0.014). The meta-analysis also revealed that in both European and Asian populations, the CLTA-4 +49 G allele was associated with the risk of RA. These results suggested that the CTLA-4 gene might be involved in the susceptibility to RA in the Chinese Han population and both +49 and CT60 of CTLA-4 gene might be the causal variants in RA disease.

Genetically modified dendritic cell (DC) vaccines expressing tumor-associated antigens are currently used for cancer immunotherapy. Peripheral blood (PB) monocyte precursors are a relatively convenient source of DCs for use in clinical studies, but are often contaminated by lymphocytes. The current study was conducted to examine the impact of T-lymphocyte contamination on genetically modified DC product. PB monocyte-derived DCs were efficiently transduced (75-95%) with an HIV-1-based self-inactivating lentiviral vector encoding a model antigen, the enhanced green fluorescent protein (eGFP). The lymphocyte-free DC culture transduced with Lenti-eGFP showed stable expression of eGFP without measurable decline in viability. In contrast, the eGFP-positive DCs disappeared rapidly in transduced DC cultures containing lymphocyte contaminants, concurrent with detectable activation and expansion of T-lymphocytes. Upon antigen recall, these T cells elicited major histocompatability complex-restricted antigen-specific cytotoxicity against eGFP-positive autologous DCs and mitogen-stimulated T lymphoblasts, mainly through the perforin-mediated pathway. In summary, this study demonstrate that the relative purity of DC cultures could determine the persistence of gene-modified DC, which may affect the induction of effective immune responses by DC vaccination strategies.

We previously demonstrated that 7-d-old rat pups exposed to hypoxia from birth exhibit ACTH-independent increases in corticosterone associated with an increase in steroidogenic acute regulatory (StAR) and peripheral-type benzodiazepine receptor (PBR) proteins. The purpose of the present study was to determine whether this increase in corticosterone could be attenuated by chemical sympathectomy induced with guanethidine treatment. Rat pups were exposed to normoxia or hypoxia from birth and treated with vehicle or guanethidine and studied at 7 d of age. Hypoxia per se resulted in an increase in plasma corticosterone without a change in plasma ACTH. Guanethidine treatment attenuated the increase in basal corticosterone in hypoxic pups but did not attenuate ACTH-stimulated corticosterone production. This effect was specific as basal and ACTH-stimulated aldosterone was not affected. Guanethidine also attenuated the increase in StAR protein induced by hypoxia. Neither the effect of hypoxia nor that of guanethidine could be explained by changes in the levels of adrenal tyrosine hydroxylase, StAR, or P450scc mRNA, adrenal tyrosine hydroxylase immunohistochemistry, or adrenal catecholamine content. We conclude that chemical sympathectomy normalizes basal corticosterone levels but has no effect on ACTH-stimulated corticosterone levels in 7-d-old rats exposed to hypoxia from birth. The mechanism of the effect of guanethidine to normalize hypoxia-stimulated basal corticosterone remains to be identified, although StAR protein may be an important mediator. This ACTH-independent increase in corticosterone may be a mechanism by which the neonate can increase circulating glucocorticoids necessary for survival while bypassing the hyporesponsiveness of the neonatal hypothalamic-pituitary-adrenal axis.

The adrenocortical response to hypoxia may be a critical component of the adaptation to this common neonatal stress. Little is known about adrenal function in vivo in hypoxic neonates. The purpose of this study was to evaluate adrenocortical responses to ACTH in suckling rat pups exposed to hypoxia from birth to 5-7 days of age compared with normoxic controls. We also evaluated potential cellular controllers of steroidogenic function in situ. In 7-day-old pups at 0800, hypoxia from birth resulted in increased basal (12.2 +/- 1.4 ng/ml; n = 12) and ACTH-stimulated (94.0 +/- 9.4 ng/ml; n = 14) corticosterone levels compared with normoxic controls (basal = 8.3 +/- 0.5 ng/ml; n = 11; stimulated = 51.3 +/- 3.8 ng/ml; n = 8). This augmentation occurred despite no significant difference in plasma ACTH levels in normoxic vs. hypoxic pups before (85 +/- 4 vs. 78 +/- 8 pg/ml) or after (481 +/- 73 vs. 498 +/- 52 pg/ml) porcine ACTH injection (20 microg/kg). This effect was similar in the afternoon at 6 days of age and even greater at 5 days of age at 0800. The aldosterone response to ACTH was not augmented by exposure to hypoxia from birth. Adrenocortical hypoxia-inducible factor (HIF)-1alpha mRNA was undetectable by RT-PCR. Steroidogenic acute regulatory (StAR) protein in adrenal subcapsules (zona fasciculata/reticularis) was augmented by exposure to hypoxia; this effect was greatest at 5 days of age. Peripheral-type benzodiazepine receptor (PBR) protein was also increased at 6 and 7 days of age in pups exposed to hypoxia from birth. We conclude that hypoxia from birth results in an augmentation of the corticosterone but not aldosterone response to ACTH. This effect appears to be mediated at least in part by an increase in controllers of mitochondrial cholesterol transport (StAR and PBR) and to occur independently of measurable changes in endogenous plasma ACTH. The augmentation of the corticosterone response to acute increases in ACTH in hypoxic pups is likely to be an important component of the overall physiological adaptation to hypoxia in the neonate.

Hypoxia from birth results in a decrease in body weight gain, body size, and bone mineral density (BMD). The purpose of the present study was to determine whether short-term administration of growth hormone (GH) (rat GH; 100 microg/d) could attenuate some of these effects of neonatal hypoxia. Rat pups (with their lactating dams) were exposed to hypoxia (vs normoxic control) from birth. Hypoxia was continued until 14 d of age, with rat GH (vs vehicle control) administered daily. Hypoxia significantly inhibited body weight gain; GH therapy did not reverse this effect. GH therapy did reverse the inhibitory effect of hypoxia on tail length but not on body length. Hypoxia decreased BMD analyzed by dual X-ray absorptiometry (DXA); this effect was not reversed by GH therapy. Both GH therapy and hypoxia decreased the percentage of body fat analyzed by DXA, the effects of which were additive when combined. There were minimal effects of hypoxia and GH therapy on plasma insulin-like growth factor-1 (IGF-1), IGF-binding protein-3, and hepatic IGF-1 mRNA expression. We conclude that some of the effects of hypoxia on body habitus are reversed by GH therapy, but that short-term GH therapy did not prevent a loss of BMD. GH therapy for more than 14 days may be necessary to appreciate fully its potential in the treatment of the sequelae of neonatal hypoxia.

We have recently identified a novel transcript of the CTLA-4 gene that may represent a native soluble form of CTLA-4 (sCTLA-4). To determine whether sCTLA-4 was expressed in humans, we applied a sensitive enzyme immunoassay on serum from patients with autoimmune thyroid disease (ATD). Eleven of 20 patients with ATD had circulating levels of sCTLA-4 ranging from 28 to 78 ng/ml, whereas only 1 of 30 apparently healthy volunteers had a level greater than 4 ng/ml. sCTLA-4 immunoreactivity was inhibited by its binding to B7.1, suggesting that sCTLA-4 is a functional receptor. Immunoprecipitation analysis of serum from patients with ATD revealed a polypeptide consistent with the predicted size of sCTLA-4. We conclude that a native soluble form of CTLA-4 is derived from an alternate transcript of the CTLA-4 gene, and its level in plasma is elevated among a population of patients with ATD.

CTLA-4 is an immunoregulatory receptor expressed on the surface of activated T and B lymphocytes. The counterreceptors for CTLA-4 are the B7 family molecules. We describe alternatively spliced mRNAs expressed in hematolymphoid tissues of humans, mice, and rats that lack the transmembrane domain coded by exon 3 of the CTLA-4 gene. These alternate transcripts were detected by RT-PCR in B cells and resting T cells of both the CD4 and the CD8 phenotype. Activation of human blood mononuclear cells with PHA or anti-CD3 + anti-CD28 monoclonal antibodies appears to effect a decrease in the amount of the alternative transcript relative to the full-length transcript. Recombinant sCTLA-4 is a B7-binding protein and has immunomodulatory effects as measured by inhibition of the mixed leukocyte response. Human serum contains immunoreactive material consistent with a native soluble form of CTLA-4., (Copyright 2000 Academic Press.)

Adaptation to hypoxia in the neonate requires an appropriate adrenocortical response. The purpose of this study was to examine the adaptation of the aldosterone pathway in rat pups exposed to hypoxia in vivo from birth to 7 days of age. Neonatal rats (with their lactating dams) were exposed to normoxia (21% O2) or hypoxia (12% O2) continuously for 7 days from birth. Trunk blood was collected, and entire adrenal glands were processed from 7-day-old rats to study the activity of the steroidogenic pathway in dispersed cells and isolated mitochondria, for measurement of expression of the steroidogenic enzyme messenger RNAs (mRNAs) by RT-competitive PCR and in situ hybridization histochemistry, for measurement of zona glomerulosa width by immunohistofluorescent staining for P450c11AS protein, and for measurement of mitochondrial number and distribution by transmission electron microscopy. Exposure to hypoxia for 7 days from birth resulted in a marked increase in plasma ACTH, corticosterone, and aldosterone with no change in PRA. Aldosteronogenesis and P450c11AS activity were both augmented in dispersed cells; this effect was lost in isolated mitochondria (from entire adrenal glands) using a permeable substrate for P450c11AS. There was no significant effect of hypoxia on expression of the steroidogenic enzyme mRNAs measured by RT-competitive PCR or in situ hybridization histochemistry. Finally, hypoxia had no effect on mitochondrial number or stereology as assessed by transmission electron microscopy or on zona glomerulosa width as assessed by staining for P450c11AS protein. We conclude that, as opposed to that in adults, hypoxia in the neonate results in an augmentation of aldosteronogenesis. This effect is not accounted for by a change in steroidogenic enzyme mRNA expression, zona glomerulosa width (i.e. hyperplasia), or mitochondrial number or distribution. This functional augmentation of aldosteronogenesis may be due to a change in mitochondrial permeability to steroid substrates and/or the effect of cytosolic factors that control mitochondrial steroidogenesis.

This article describes the development of a new experimental model using rats for the evaluation of small-caliber vascular grafts. By modifying heterotopic heart transplantation, two 1.5- to 2.0-cm long vascular prostheses were interposed between a syngeneic donor heart and the recipient abdominal vessels in the form of vascular bridges. Once blood flow through the vascular grafts was reestablished, the donor heart resumed normal beating. The status of the vascular grafts could be easily monitored by palpation. Occlusion of the grafts stopped donor heart beating without affecting survival of the animals. Once the surgical method was mastered, the postoperative mortality was approximately 10%, and the total procedure took less than 2 hours. Although microvascular surgical technique and equipment are required, this model has several advantages, including easy detection of thrombotic occlusion of the grafts, the use of small animals of defined genetic background, the absence of effect of graft occlusion on the recipient's life, and possible repeated operation on the same animal.

Rat strains with congenitally reduced total hemolytic complement activity do not reject cardiac xenografts hyperacutely. Prolongation of graft survival in the guinea pig-to-C6-deficient PVG rat donor/recipient combination has been observed. However, experience with this model has been complicated by a high postoperative mortality from respiratory distress. The authors hypothesized that placement of the xenograft resulted in local activation of complement, which contributed to remote pulmonary injury leading to respiratory dysfunction. To test this hypothesis, an attempt was made to reduce early complement component activation with the use of an antibody to rat C3 in C6-deficient PVG recipients. Six of eight untreated C6-deficient PVG recipients died in the immediate postoperative period with vigorously beating heart grafts, whereas only 2 of 14 C6-deficient recipients pretreated with anti-rat C3 antibody died within 24 h postoperatively. Although pretreatment with anti-C3 antibody improved survival of recipients, the duration of cardiac xenograft survival was similar whether the recipients were pretreated or not. The use of anti-C3 antibody in C6-deficient rats is a valid approach to studying xenotransplantation in the absence of hyperacute rejection and has an additional advantage in that it does not require the use of expensive reagents such as cobra venom factor.

Hypoxia leads to a decrease in aldosterone that cannot be entirely explained by extrinsic controllers of adrenal function. We have shown that acute hypoxia attenuates aldosterone synthesis via a direct inhibition of the function of the aldosterone enzyme pathway. The mechanism of the sustained decrease in aldosterone during chronic hypoxia is unknown. The present study evaluated the hypothesis that chronic hypoxia leads to a decrease in the expression of the steroidogenic enzyme P-450c11AS unique to the aldosterone pathway. Rats were exposed to 3 days of normoxia, moderate hypoxia (12% O2), or severe hypoxia (10% O2). Adrenal glands were removed and prepared for biochemical analysis of steroidogenesis in vitro (dispersed capsular cells) and for measurement of steady-state enzyme mRNA levels by reverse-transcription competitive polymerase-chain reaction (RT-cPCR) and by in situ hybridization histochemistry (ISHH). Moderate hypoxia had no effect on steroidogenesis. Adrenal cells from rats exposed to severe hypoxia demonstrated a decreased conversion of corticosterone to aldosterone (late pathway catalyzed by P-450c11AS) without a change in the other mitochondrial cytochrome P-450 enzyme activities. Adrenal cells from rats exposed to hypoxia also demonstrated a three- to fourfold decrease in P-450c11AS mRNA without a change in the other mitochondrial cytochrome P-450 enzymes mRNAs, as determined by either RT-cPCR or ISHH. We conclude that relatively short-term chronic hypoxia in rats leads to a decrease in aldosteronogenesis by decreasing the expression of the gene for the late-pathway enzyme unique to the aldosterone pathway (P-450c11AS).

We used reverse transcription-polymerase chain reaction (RT-PCR) to clone a rat complementary DNA that encoded the PVG rat granulocyte-macrophage colony-stimulating factor (GM-CSF). PCR products were cloned into a eukaryotic expression vector and transfected into the mouse myeloma cell line Sp2/0-Ag14. Cell culture supernatants of two of these transfectants supported proliferation of the growth factor-dependent cell line, DA-3, and promoted myeloid colony formation in rat and mouse bone marrow cell (BMC) cultures. The GM-CSF activity in these supernatants was neutralized by a polyclonal antibody to mouse GM-CSF. The cloning and expression of rat GM-CSF provides a valuable reagent for the study of the biology and clinical applications of the GM-CSFs.

The adrenocortical enzymes of the steroidogenic late pathway in the rat are aldosterone synthase (P450aldo), which catalyzes the production of aldosterone, and 11 beta-hydroxylase (P45011 beta), which catalyzes the production of corticosterone throughout the cortex. These two enzymes are highly homologous and are encoded by the genes CYP11B2 and CYP11B1, respectively. The purpose of the present study is to describe the development of two sets of primers and the reverse transcription-polymerase chain reaction (RT-PCR) conditions that are capable of discriminating between rat P450aldo and P45011 beta mRNAs. The P450aldo primer set did not amplify full length cDNA P45011 beta plasmid and the P45011 beta primer set did not amplify full length cDNA P450aldo plasmid indicating minimal crosstalk. The fidelity of the PCR primers and method was further established by sequencing the PCR products and demonstration of virtual identity with the published sequences of P450aldo and P45011 beta. RT-PCR of mRNA from adrenal capsules (zona glomerulosa) and subcapsules (zona reticularis/fasciculata) from rats demonstrated no effect of sodium diet on the expression of P45011 beta mRNA but an approximately 8-fold greater expresison in P450aldo mRNA on low vs high sodium intake. Similar results were found when single hemicapsules were subjected to RT-PCR, demonstrating the sensitivity of the method. We conclude that the two sets of PCR primers and the RT-PCR method described are capable of evaluating the expression of the highly homologous mRNAs for P450aldo and P45011 beta with great precision and sensitivity.

Intragraft T-cell receptor (TCR) alpha and beta chain variable region gene expression was analyzed in human cardiac allograft biopsies by reverse transcription polymerase chain reaction. Rearranged TCR alpha and beta chain gene transcripts were detected in all biopsies examined (N = 23), indicating the presence of T cells bearing the alpha/beta TCR even in the absence of microscopically apparent leukocyte infiltration. In this analysis, a broad TCR alpha/beta repertoire in actively rejecting lesions was demonstrated, whereas fewer TCR alpha and beta chain gene families were detected in nonrejecting lesions. The number of expressed TCR V beta chain gene families typically was two- to sixfold higher than that of V alpha chain families in all biopsies tested. This asymmetric relation was present throughout the histologic grading spectrum of the biopsies. Based on these data, the TCR repertoire is heterogenous even in the early stages of mononuclear cell infiltration of the allograft. Also based on the data, the presence of T cells in grafts with minimal cellular infiltrates is not a specific marker of subsequent rejection episode, because T cells were identified in all allograft biopsies.

We have defined the chromosomal location of the human MAGE (melanoma antigen) gene family by polymerase chain reaction amplification of several segments of the MAGE genes from somatic cell hybrids containing well-defined groups of human chromosomes and hybrids containing human derivative chromosomes. The data show that the three known MAGE family members (MAGEs -1, -2, & -3) are syntenic and map to the human X chromosome region q27-qter. Because males and females are hemizygous for most X-linked genes, the frequency of antigen-loss variants for the MAGE system is expected to be greater in comparison to antigens encoded by somatic chromosomes. In this regard, we believe that patients enrolled in MAGE-specific immunotherapy trials should be carefully monitored for the presence of MAGE antigen-loss variants.

The effects of cimetidine, an H2 receptor antagonist, on subpopulation of splenic lymphocytes were studied in mice. Cimetidine (50 mg/kg) was given to groups of mice by intraperitoneal injection. The splenic mononuclear cells from treated and control animals were evaluated for relative number of lymphocyte subpopulations (i.e. LYT1, LYT2, L3T4 and GAM cells) by flow cytometry. The percentages of LYT2 cells (suppressor equivalent) were significantly lower (5.5 vs 12.0%) in mice who were treated with cimetidine vs control animals. The percentages of L3T4 (helper equivalent) were not statistically different in any of the experimental groups.

A case of pre-B cell acute lymphoblastic leukemia (pre-B ALL) with a dir ins(11;4)(q23;q21q31) chromosome rearrangement is presented. The patient's clinical findings and history were similar to those described for the t(4;11)(q21;q23) subgroup of childhood ALL. These findings suggest that the interfacing of the distal breakpoint at band 4q21 to the proximal breakpoint of band 11q23 represents the primary cytogenetic change observed in this subgroup of ALL.

We have used a variety of standard inbred, recombinant, and congenic rat strains to establish the effect of MHC and non-MHC genetic incompatibilities on bone marrow transplantation. The role of these loci in the successful establishment of bone marrow engraftment was first determined by examining the potential of donor marrow to protect recipient rats from a lethal dose of the myeloablative drug busulfan. Similar donor/recipient combinations were then used to examine the effects of the same incompatibilities on the induction of fatal graft-versus-host disease (GVHD) in recipients conditioned with a combination of busulfan and cyclophosphamide. The data obtained indicate that: (1) a difference for the entire MHC of the rat is sufficient to prevent marrow engraftment and to produce fatal GVHD; (2) the class I RT1.A locus of the rat MHC has a differential effect on bone marrow transplantation, since disparity for this locus prevents successful marrow engraftment, while this gene has little effect on the development of fatal GVHD; (3) disparity for the class I RT1.E locus has no effect on bone marrow engraftment and does not stimulate GVHD; (4) disparity for the class II locus, RT1.D, can prevent marrow engraftment and elicit fatal GVHD; and (5) incompatibility for non-MHC genes can prevent the establishment of bone marrow engraftment and elicit fatal GVHD.

A disease similar to the chronic graft-versus-host disease (cGVHD) seen following transplantation of human bone marrow was observed after syngeneic and allogeneic bone marrow transplantation in rats. Bone marrow grafts were exchanged between donors and recipients that were syngeneic or genetically different for the RT2 erythrocyte antigen locus by the use of AUG/AUG.2B and PVG/PVG.2A congenic pair donor/recipient strain combinations. After an initial period of well-being (120-180 days posttransplantation), several AUG and AUG.2B recipients of syngeneic or RT2-mismatched bone marrow developed clinical signs compatible with cGVHD. The clinical signs of the disease included: erythema, diffuse alopecia, thickened skin folds, and conjunctivitis. Laboratory findings included peripheral blood eosinophilia and impaired in-vitro proliferative responses to third-party spleen cells in the mixed lymphocyte reaction. Histological examination of the tissues of a limited number of rats with cGVHD showed subepidermal mononuclear inflammation with atrophy of the epidermis and adnexa of the skin, as well as plasmacytic hyperplasia of the lymphoid tissues. None of the PVG or PVG.2A recipients of syngeneic or RT2-mismatched marrow developed cGVHD. The development of cGVHD in AUG.2B recipients of syngeneic marrow and the absence of the disease in reciprocal marrow grafts between the PVG/PVG.2A rat strains suggests that the development of the disease in the AUG and AUG.2B recipients of RT2-mismatched bone marrow is not due to the RT2 disparity, but may be due to an autologous immune reaction. Furthermore, the finding that the cGVHD is only observed when the AUG and AUG.2B strains are used as recipients--not when the PVG or PVG.2A strains are used as recipients--suggests that the development of the disease is associated with the genetic background of the host and is independent of the background of the donor. It is possible that the use of high-dose cyclophosphamide treatment is involved in the pathogenesis of cGVHD, because the disease is observed only when the recipients are conditioned for transplantation with this immunosuppressive agent.