Early expression of mature αß TCR in CD4-CD8- T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations.

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αß T cell antigen receptor (TCR). We report that these early αß TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αß T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αß TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

A novel method for analysis of human T cell repertoires by real-time PCR.

T lymphocyte responses to challenges with multiple pathogens depend on the diversity of their T cell receptors (TcRs) that are heteroduplexes of alpha and beta chains. The regions of alpha and beta chains that define TcR specificity are encoded by rearranged variable (V) and joining (J) genes that are separated by variable numbers of nucleotides that encode the complementarity determining region 3 (CDR3). The assumption that a "healthy" T cell compartment exhibits broad TcR and CDR3 diversity has driven development of methods to evaluate diversity of TcR beta transcripts expressed by T lymphocyte populations and subpopulations in inflammatory sites and human malignancies. To that end, we have developed the BV:BJ matrix assay that uniquely generates a single statistic that describes TcR repertoire diversity and improves identification of beta transcripts expressed by expanded T cell clonotypes. The BV:BJ matrix uses rigorously selected primers specific for individual V and J genes to amplify beta transcripts in real-time PCRs driven by 533 BV:BJ primer pairs. The quantitative control of real-time PCRs produces Shannon entropy estimates of diversity that are reproducible over a range of template amounts and amenable to statistical analyses that have been difficult to perform with existing methods of repertoire analysis.

In utero transplanted human hepatocytes allow postnatal engraftment of human hepatocytes in pigs.

In utero cell transplantation (IUCT) can lead to the postnatal engraftment of human cells in the xenogeneic recipient. Most reports of IUCT have involved hematopoietic stem cells. It is unknown whether human hepatocytes used for IUCT in fetal pigs will lead to the engraftment of these same cells in the postnatal environment. In this study, fetal pigs received direct liver injections of 1 × 10(7) human hepatocytes in utero and were delivered by cesarean section at term. The piglets received a second direct liver injection of 5 × 10(7) human hepatocytes 1 week after birth. The serum was analyzed for human albumin 2, 4, and 6 weeks after engraftment. Piglet livers were harvested 6 weeks after transplantation and were examined by immunohistochemistry, polymerase chain reaction, and fluorescence in situ hybridization for human-specific sequences. Piglets undergoing IUCT with human hepatocytes that were postnatally engrafted with human hepatocytes showed significant levels of human albumin production in their serum at all postengraftment time points. Human albumin gene expression, the presence of human hepatocytes, and the presence of human beta-2 microglobulin were all confirmed 6 weeks after engraftment. IUCT in fetal pigs with human hepatocytes early in gestation allowed the engraftment of human hepatocytes, which remained viable and functional for weeks after transplantation. IUCT followed by postnatal engraftment may provide a future means for large-scale expansion of human hepatocytes in genetically engineered pigs.

The anticancer drug Dp44mT inhibits T-cell activation and CD25 through a copper-dependent mechanism.

The di-2-pyridylketone thiosemicarbazone Dp44mT is a metal-chelating compound that has been demonstrated to have potent activity as an anticancer agent. Here we report that it also has a dramatic inhibitory effect on T-cell activation in vitro. We found that 10 nM Dp44mT (IC(50) 3.2 nM) prevented the up-regulation of surface CD25, and completely suppressed the activation and proliferation of splenic T cells isolated from Mus musculus that were stimulated with either T-cell receptor (TCR) cross-linking antibodies or phorbol ester plus ionomycin. In contrast, Dp44mT had no adverse effects on the survival of resting T cells. In addition, T cells stimulated in the presence of Dp44mT maintained the ability to up-regulate CD69 surface expression and secrete interleukin-2. Consistent with these observations, Dp44mT did not inhibit multiple canonical signals downstream of the TCR, including the nuclear factor of activated T cells. The effects of Dp44mT were easily mitigated by addition of nontoxic copper chelators or N-acetylcysteine, indicating a role for copper and reactive oxygen species in its actions. Together, these findings suggest that Dp44mT may serve as a potent immunosuppressive agent that could complicate its use as a cancer therapeutic agent, but might have utility in the treatment of autoimmunity.

A profile of immune response to herpesvirus is associated with radiographic joint damage in rheumatoid arthritis.

INTRODUCTION: Progression of joint damage despite appropriate therapy remains a significant problem for patients with rheumatoid arthritis (RA). This study was undertaken to identify profiles of immune response that correlate with radiographic joint damage as a first step toward the discovery of new pathogenic mechanisms of joint destruction in RA. METHODS: The study included 58 patients with RA and 15 healthy controls. The profiles of cytokine release from peripheral blood mononuclear cells (PBMC) in response to stimulation for 48 hours with one of six stimuli, or in media alone, were measured. Immune response profiles identified for each stimulus were correlated with radiographic joint damage as defined by the Sharp-van der Heijde score (SHS), before and after multivariable adjustment. For profiles correlated with the SHS, the distributions of individual cytokines were evaluated in patients according to the severity of joint damage and compared to healthy controls. RESULTS: The immune response profile for cytomegalovirus (CMV)/Epstein-Barr virus (EBV) stimulation was correlated with both the SHS total and erosion scores (r = 0.31, P = 0.018 and r = 0.33, P = 0.011, respectively). After adjusting for age, sex, disease duration, autoantibody status, CMV/EBV serological status, current disease activity, disability and treatments, the correlation of the CMV/EBV immune response and the SHS erosion score became stronger (r = 0.43, P < 0.003). The CMV/EBV immune response correlated with CMV IgG (r = 0.44, P < 0.001), but not with EBV IgG. The most important cytokines for the CMV/EBV immune response profile were IFN-γ, IL-2, IL-4, IL-5, IL-13 and IL-17A, all of which are associated with T-cell immunity. Both the summary immune response score and the individual responses of IFN-γ and IL-13 to CMV/EBV stimulation were associated with greater joint damage. CONCLUSIONS: A profile of immune response to purified CMV/EBV lysates is associated with radiographic joint damage. The correlation of this immune response to CMV serology implies possible involvement of latent CMV infection. Therefore, the findings suggest that the immune response to latent CMV infection could play a fundamental role in the progression of inflammation and structural joint damage in patients with RA.

Does lung ischemia and reperfusion have an impact on coronary flow? A quantitative coronary blood-flow analysis with inflammatory cytokine profile.

OBJECTIVE: Ischemia-reperfusion (IR) injury remains a major cause of early morbidity and mortality after lung transplantation with poorly documented extrapulmonary repercussions. To determine the hemodynamic effect due to lung IR injury, we performed a quantitative coronary blood-flow analysis in a swine model of in situ lung ischemia and reperfusion. METHODS: In 14 healthy pigs, blood flow was measured in the ascending aorta, left anterior descending (LAD), circumflex (Cx), right coronary artery (RCA), right common carotid artery (RCCA), and left internal mammary artery (LIMA), along with left-and right-ventricular pressures (LVP and RVP), aortic pressure (AoP), and pulmonary artery pressure (PAP). Cardiac Troponin (cTn), interleukin 6 and 10 (IL-6 and IL-10), and tumor necrosis factor A (TNF-A) were measured in coronary sinus blood samples. The experimental (IR) group (n=10) underwent 60 min of lung ischemia followed by 60 min of reperfusion by clamping and releasing the left pulmonary hilum. Simultaneous measurements of all parameters were made at baseline and during IR. The control group (n=4) had similar measurements without lung IR. RESULTS: In the IR group, total coronary flow (TCF=LAD+Cx+RCA blood-flow) decreased precipitously and significantly from baseline (113±41 ml min"1) during IR (p<0.05), with the lowest value observed at 60 min of reperfusion (-37.1%, p<0.003). Baseline cTn (0.08±0.02 ng ml(-1)) increased during IR and peaked at 45 min of reperfusion (+138%, p<0.001). Baseline IL-6 (9.2±2.17 pg ml(-1)) increased during IR and peaked at 60 min of reperfusion (+228%, p<0.0001). Significant LVP drop at 5 min of ischemia (p<0.05) was followed by a slow return to baseline at 45 min of ischemia. A second LVP drop occurred at reperfusion (p<0.05) and persisted. Conversely, RVP increased throughout ischemia (p<0.05) and returned toward baseline during reperfusion. Coronary blood flow and hemodynamic profile remained unchanged in the control group. IL-10 and TNF-A remained below the measurable range for both the groups. CONCLUSIONS: In situ lung IR has a marked negative impact on coronary blood flow, hemodynamics, and inflammatory profile. In addition, to the best of our knowledge, this is the first study where coronary blood flow is directly measured during lung IR, revealing the associated increased cardiac risk.

MHC class II epitope nesting modulates dendritic cell function and improves generation of antigen-specific CD4 helper T cells.

CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.

A degenerate HLA-DR epitope pool of HER-2/neu reveals a novel in vivo immunodominant epitope, HER-2/neu88-102.

PURPOSE: Over the past two decades, there has been significant interest in targeting HER-2/neu in immune-based approaches for the treatment of HER-2/neu+ cancers. For example, peptide vaccination using a CD8 T cell-activating HER-2/neu epitope (amino acids 369-377) is an approach that is being considered in advanced phase clinical trials. Studies have suggested that the persistence of HER-2/neu-specific CD8 T cells could be improved by incorporating human leukocyte antigen (HLA) class II epitopes in the vaccine. Our goal in this study was to identify broad coverage HLA-DR epitopes of HER-2/neu, an antigen that is highly expressed in a variety of carcinomas. EXPERIMENTAL DESIGN: A combination of algorithms and HLA-DR-binding assays was used to identify HLA-DR epitopes of HER-2/neu antigen. Evidence of preexistent immunity in cancer patients against the identified epitopes was determined using IFN-gamma enzyme-linked immunosorbent spot (ELIspot) assay. RESULTS: Eighty-four HLA-DR epitopes of HER-2/neu were predicted, 15 of which had high binding affinity for > or =11 common HLA-DR molecules. A degenerate pool of four HLA-DR-restricted 15-amino acid epitopes (p59, p88, p422, and p885) was identified, against which >58% of breast and ovarian cancer patients had preexistent T-cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 84% of population. Lastly, in this degenerate pool, we identified a novel in vivo immunodominant HLA-DR epitope, HER-2/neu(88-102) (p88). CONCLUSION: The broad coverage and natural immunity to this epitope pool suggests potential usefulness in HER-2/neu-targeting, immune-based therapies such as vaccines.

Identification of a broad coverage HLA-DR degenerate epitope pool derived from carcinoembryonic antigen.

CD4 T cells are important for anti-tumor immune responses. Aside from their role in the activation of CD8 T cells, CD4 T cells also mediate anti-tumor immune responses by recruiting innate immune effectors into the tumor microenvironment. Thus, the search for strategies to boost CD4 T cell immunity is an active area of research. Our goal in this study was to identify HLA-DR epitopes of carcinoembryonic antigen (CEA), a commonly over-expressed tumor antigen. HLA-DR epitopes of CEA were identified using the epitope prediction program, PIC (predicted IC(50)) and tested using in vitro HLA-DR binding assays. Following CEA epitope confirmation, IFN-gamma ELIspot assays were used to detect existing immunity against the HLA-DR epitope panel of CEA in breast and ovarian cancer patients. In vitro generated peptide-specific CD4 T cells were used to determine whether the epitopes are naturally processed from CEA protein. Forty-three epitopes of CEA were predicted, 15 of which had high binding affinity for 8 or more common HLA-DR molecules. A degenerate pool of four, HLA-DR restricted 15 amino acid epitopes (CEA.24, CEA.176/354, CEA.488, and CEA.653) consisting of two novel epitopes (CEA.24 and CEA.488) was identified against which 40% of breast and ovarian cancer patients had pre-existent T cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 94% of patients. Patients with breast or ovarian cancer demonstrate pre-existent immune responses to the tumor antigen CEA. The degenerate pool of CEA peptides may be useful for augmenting CD4 T cell immunity.

Beneficial effect of TRAIL on HIV burden, without detectable immune consequences.

BACKGROUND: During uncontrolled HIV disease, both TNF-related apoptosis inducing ligand (TRAIL) and TRAIL receptor expression are increased. Enhanced TRAIL sensitivity is due to TRAIL receptor up-regulation induced by gp120. As a result of successful antiretroviral therapy TRAIL is down-regulated, and there are fewer TRAIL-sensitive cells. In this setting, we hypothesized that all cells that contain virus, including those productively- and latently-infected, have necessarily been "primed" by gp120 and remain TRAIL-sensitive, whereas uninfected cells remain relatively TRAIL-resistant. METHODS AND FINDINGS: We evaluated the immunologic and antiviral effects of TRAIL in peripheral blood lymphocytes collected from HIV-infected patients with suppressed viral replication. The peripheral blood lymphocytes were treated with recombinant TRAIL or an equivalent amount of bovine serum albumin as a negative control. Treated cells were then analyzed by quantitative flow cytometry, ELISPOT for CD4+ and CD8+ T-cell function, and limiting dilution microculture for viral burden. Alterations in the cytokine milieu of treated cells were assessed with a multiplex cytokine assay. Treatment with recombinant TRAIL in vitro reduced viral burden in lymphocytes collected from HIV-infected patients with suppressed viral load. TRAIL treatment did not alter the cytokine milieu of treated cells. Moreover, treatment with recombinant TRAIL had no adverse effect on either the quantity or function of immune cells from HIV-infected patients with suppressed viral replication. CONCLUSIONS: TRAIL treatment may be an important adjunct to antiretroviral therapy, even in patients with suppressed viral replication, perhaps by inducing apoptosis in cells with latent HIV reservoirs. The absence of adverse effect on the quantity or function of immune cells from HIV-infected patients suggests that there is not a significant level of "bystander death" in uninfected cells.

An HLA-DR-degenerate epitope pool detects insulin-like growth factor binding protein 2-specific immunity in patients with cancer.

Recent studies have shown the importance of helper CD4 T cells in initiating and sustaining tumor-specific CD8 T-cell immunity. This has paved the way for identifying MHC class II epitopes that could be incorporated into class I-based vaccines. In this study, the goal was to identify an HLA-DR-degenerate epitope pool derived from insulin-like growth factor binding protein 2 (IGFBP-2). IGFBP-2, a regulator of insulin-like growth factor action, is overexpressed in the majority of breast and ovarian cancers. Using algorithms, we predicted 29 HLA-DR1-binding epitopes. Binding assays targeting 15 different HLA-DRs revealed that 10 epitopes were degenerate, binding to at least four different HLA-DR variants. An IFN-gamma enzyme-linked immunosorbent spot assay was used to assess immunity to these 10 epitopes in 48 patients with either breast or ovarian cancer and 18 controls. Elevated T-cell immunity in patients was detected in 4 of the 10 epitopes (IGFBP2.17, IGFBP2.22, IGFBP2.249, and IGFBP2.293). The cumulative T-cell frequency of these four epitopes was elevated in patients relative to controls. All four peptides are naturally processed and presented to CD4 T-cells. The degenerate pool of peptides covers nearly 80% of patients and may be useful for augmenting CD4 T-cell immunity in patients undergoing immunization.

T-cell immunity to the folate receptor alpha is prevalent in women with breast or ovarian cancer.

PURPOSE: Studies have demonstrated that the generation of immunity to tumor antigens is associated with improved prognosis for many cancers. A candidate antigen is the folate receptor alpha (FRalpha), which is overexpressed in breast and ovarian cancers. Our goal in this study was to attain a better understanding of the extent of endogenous FRalpha immunity. METHODS: Using a CD4+ T cell epitope prediction algorithm, we predicted promiscuous epitopes of FRalpha, and tested for immunity in 30 breast (n = 17) or ovarian (n = 13) cancer patients and 18 healthy donors using enzyme-linked immunospot analysis. RESULTS: Fourteen peptides were predicted, seven each from the carboxy- and amino-terminus halves of the protein. More than 70% of patients demonstrated immunity to at least one FRalpha peptide. Patients responded to an average of 3 +/- 0.5 peptides, whereas healthy donors responded to 1 +/- 0.4 peptides (P = .004). Five peptides were recognized by more than 25% of patients. Responses to three peptides were higher (P < .05) in patients than in healthy donors, suggesting augmented immunity. Compared with healthy individuals, patients developed higher immunity to the amino-terminus half of the receptor (P = .03). There was no difference between each group in the responses to nonspecific (P = .2) and viral stimuli (P = .5). Lastly, patients demonstrated elevated levels of FRalpha antibodies consistent with a coordinated immune response. CONCLUSION: These findings demonstrate that the FRalpha is a target of the immune system in breast and ovarian cancer patients. Understanding which antigens are targeted by the immune system may be important for prognosis or immune-based therapies.

Expression of mRNA for a newly identified Pax5 exon is reduced in multiple myeloma.

Pax5 is a transcription factor that is critical in the bone marrow for differentiation and proliferation of B cells until the plasma cell stage. In Pax5(-/-) mice, B-cell development stalls at the pro-B-cell stage. Messenger RNA profiles of alternatively spliced isoforms of Pax5 in bone marrow frequently differ between multiple myeloma (MM) patients and healthy donors. We sought to determine if Pax5 mRNA profiles also differed in blood and unexpectedly detected the presence of a previously unreported exon that alters the amino acid code for the transactivating domain of Pax5 in CD138(-) B cells. This unique exon escapes detection by conventional analyses of RT-PCR products and may serve as a prototype for other exons, in other genes, that escape RT-PCR detection. Eight percent of tested human subjects were heterozygous for an allele with a nonsynonymous nucleotide substitution in the new exon, and one MM patient was homozygotic for this base difference. Subsequent analysis of plasma and B-cell populations from bone marrow revealed a markedly reduced mRNA expression of the new isoform in cells from MM patients when compared to cells from normal subjects.

Cysteine-tailed class I-binding peptides bind to CpG adjuvant and enhance primary CTL responses.

Immunostimulatory CpG motifs in synthetic oligonucleotides can be effective adjuvants for the priming of CTLs. We first observed that a single male-specific peptide (KCSRNRQYL) (HY2) was more efficient than another male-specific peptide (WMHHNMDLI) (HY1) at priming IFN-gamma-secreting CTLs in vivo when combined with lipid A and CpG and that it also visibly precipitated CpG. The addition of the six N-terminal residues (KCSRNR) from HY2 to HY1 yielded a peptide, KCSRNR-HY1, that both precipitated CpG and primed increased numbers of HY1-specific CTLs. We refer to this type of peptide as a primotope that includes a class I binding peptide tailed with amino acids that increase priming. Ala residues were substituted for the Arg/Lys residues (ACSANA-HY1), and these substitutions did not reduce in vivo priming potential. However, the substitution of Ala for Cys (KASRNR-HY1) resulted in the complete loss of priming, demonstrating the importance of Cys for in vivo priming when mixed with CpG. This result suggested that increased priming was based in disulfide bonding between Cys residues and internal phosphorothioate groups of synthetic CpG. The addition of Cys-bearing primotopes to radiolabeled CpG with a single thioate group resulted in the appearance of a new band that was inhibited by 1) Cys > Ala substitution and 2) reduction and alkylation of CpG. These results reveal a novel mechanism for complexing class I binding peptides and CpG adjuvant for development of new peptide-adjuvant combinations for vaccines for cancer and infectious diseases.

Long-term bovine hematopoietic engraftment with clone-derived stem cells.

Therapeutic cloning by somatic cell nuclear transfer offers potential for treatment of a wide range of degenerative disease. Nuclear transplantation with neo (r)-marked somatic nuclei from 10-13-year-old cows was used to generate cloned bovine fetuses. Clone fetal liver (FL) hematopoietic stem cells (HSC) were transplanted into two busulfan-treated and one untreated nuclear donor cows. Hematopoiesis was monitored over 13-16 months by in vitro progenitor and HSC assays. Chimerism was demonstrated by PCR in blood, marrow, lymph nodes, and endothelium, peaking at levels of 9-17% in blood granulocytes but at lower levels in lymphocyte subsets (0.1-0.01%). Circulating progenitors showed high levels of chimerism (up to 60% neo (r+)) with persisting fetal features. At sacrifice, the animal that had no pre-transplant myelosupression showed persisting donor cells in blood and lymph nodes, and in marrow 0.25% of progenitor cells and a detectable fraction of stem cells were neo (r+). The fetal HSC showed a 10-fold competition advantage over adult HSC. Cloning generated histocompatible HSC capable of long-term multilineage engraftment in a large animal model.

Aerosolized granulocyte macrophage colony-stimulating factor (GM-CSF) therapy in metastatic cancer.

A recent phase I study of aerosolized granulocyte macrophage colony-stimulating factor (GM-CSF) in patients with malignant metastases to the lungs demonstrated excellent tolerance and possible efficacy. This therapy was offered to other patients who refused "standard" treatment or when no effective therapy was available. Forty-five patients were treated; 40 had pulmonary metastases. Aerosolized GM-CSF (250 microg/dose) was administered twice a day using a 1 week on, 1 week off schedule. The mean interval between diagnosis and therapy was 32 months. Twenty-four patients had disease stabilization or partial regression. The mean duration of benefit was 10 months. This benefit was noted in 8 of 13 with a sarcoma, 6 of 14 with melanoma, and 5 of 12 with renal cell carcinoma. Eighteen patients reported mostly self-limiting toxicities. The frequency of certain melanoma-specific T lymphocytes in 1 patient with stable disease was found to have increased 10-fold after therapy. Aerosolized GM-CSF appears to have limited but promising efficacy in treatment of pulmonary metastatic disease. In one patient, we have evidence of upregulation of melanoma-specific cytotoxic T-cells. Further study is warranted to understand the impact of this therapy on the natural history of metastatic cancer.

Nonhuman primate parthenogenetic stem cells.

Parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Whereas parthenogenesis is a common mode of reproduction in lower organisms, the mammalian parthenote fails to produce a successful pregnancy. We herein describe in vitro parthenogenetic development of monkey (Macaca fascicularis) eggs to the blastocyst stage, and their use to create a pluripotent line of stem cells. These monkey stem cells (Cyno-1 cells) are positive for telomerase activity and are immunoreactive for alkaline phosphatase, octamer-binding transcription factor 4 (Oct-4), stage-specific embryonic antigen 4 (SSEA-4), tumor rejection antigen 1-60 (TRA 1-60), and tumor rejection antigen 1-81 (TRA 1-81) (traditional markers of human embryonic stem cells). They have a normal chromosome karyotype (40 + 2) and can be maintained in vitro in an undifferentiated state for extended periods of time. Cyno-1 cells can be differentiated in vitro into dopaminergic and serotonergic neurons, contractile cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and adipocytes. When Cyno-1 cells were injected into severe combined immunodeficient mice, teratomas with derivatives from all three embryonic germ layers were obtained. When grown on fibronectin/laminin-coated plates and in neural progenitor medium, Cyno-1 cells assume a neural precursor phenotype (immunoreactive for nestin). However, these cells remain proliferative and express no functional ion channels. When transferred to differentiation conditions, the nestin-positive precursors assume neuronal and epithelial morphologies. Over time, these cells acquire electrophysiological characteristics of functional neurons (appearance of tetrodotoxin-sensitive, voltage-dependent sodium channels). These results suggest that stem cells derived from the parthenogenetically activated nonhuman primate egg provide a potential source for autologous cell therapy in the female and bypass the need for creating a competent embryo.

Characterization of antigen processing machinery and Survivin expression in tonsillar squamous cell carcinoma.

BACKGROUND: There is a statistically significant association between human leukocyte antigen (HLA) Class I antigen expression and improved prognosis for some patients. This association reflects the control of tumor growth by HLA Class I antigen-restricted, tumor-associated antigen-specific cytolytic T cells. However, progression of other malignant diseases is not associated with the loss of HLA expression. These observations show that the poor prognosis of a subset of tumors, despite high HLA Class I antigen expression, may reflect the development of alternative mechanisms utilized by tumor cells to escape from immune recognition and destruction. METHODS: The authors evaluated the possible correlation between the expression of the antiapoptosis gene, Survivin, HLA Class I, and progression of tonsillar squamous cell carcinomas (TSCC) lesions. Tissue microarrays were constructed from primary TSCC, metastatically involved lymph nodes, adjacent normal mucosa, and tonsillar parenchyma excised for nonmalignant conditions. RESULTS: Immunoperoxidase staining of tissue sections demonstrated that Survivin expression is significantly higher (P < 0.001) in malignant tumors than in normal tissue samples. In addition, Survivin expression is significantly higher (P = 0.05) in metastatic than in primary lesions. Survivin expression in primary lesions correlated positively with delta (P = 0.025), tapasin (P = 0.028), and HLA Class I antigen (P = 0.006) expression. The expression patterns of delta, tapasin, HLA Class I antigen, beta-2-microglobulin, and Survivin did not demonstrate any significant association with the clinical course of disease. CONCLUSIONS: For TSCC that maintain the expression of HLA Class I antigen, overexpression of Survivin may provide an alternative explanation for tumor progression.

Resistance to acute babesiosis is associated with interleukin-12- and gamma interferon-mediated responses and requires macrophages and natural killer cells.

We examined the role of the cytokines gamma interferon (IFN-gamma) and interleukin-12 (IL-12) in the model of acute babesiosis with the WA1 Babesia. Mice genetically deficient in IFN-gamma-mediated responses (IFNGR2KO mice) and IL-12-mediated responses (Stat4KO mice) were infected with the WA1 Babesia, and observations were made on the course of infection and cytokine responses. Levels of IFN-gamma and IL-12 in serum increased 24 h after parasite inoculation. The augmented susceptibility observed in IFNGR2KO and Stat-4KO mice suggests that the early IL-12- and IFN-gamma-mediated responses are involved in protection against acute babesiosis. Resistance appears to correlate with an increase in nitric oxide (NO) production. In order to assess the contribution of different cell subsets to resistance against the parasite, we also studied mice lacking B cells, CD4+ T cells, NK cells, and macrophages. Mice genetically deficient in B lymphocytes or CD4+ T lymphocytes were able to mount protective responses comparable to those of immunosufficient mice. In contrast, in vivo depletion of macrophages or NK cells resulted in elevated susceptibility to the infection. Our observations suggest that a crucial part of the response that protects from the pathogenic Babesia WA1 is mediated by macrophages and NK cells, probably through early production of IL-12 and IFN-gamma, and induction of macrophage-derived effector molecules like NO.

Altered mRNA expression of Pax5 and Blimp-1 in B cells in multiple myeloma.

Multiple myeloma (MM) is a plasma cell disorder that potentially initiates during an early stage of B-cell development. We encountered an unidentified isoform of B cell-specific activator protein (BSAP, or Pax5) in MM cells while performing differential analyses to compare mRNA expression in malignant and normal plasma cells. Pax5 is a transcription factor that plays a central role throughout B-cell development until the point of terminal differentiation. Our finding of this unique isoform prompted us to investigate Pax5 isoform usage in plasma cells and B-cell populations in other MM and healthy subjects. In contrast to normal Pax5 expression, we observed multiple isoforms of Pax5 in conjunction with low levels of expression of the full-length Pax5 in B cells from MM patients. The expressed isoforms in MM varied considerably from patient to patient, with no clear pattern. We also performed semiquantitative analyses of the mRNA expression levels of B lymphocyte-induced maturation protein (Blimp-1), because expression levels of Pax5 and Blimp-1 have been shown to be inversely correlated. We observed the expression of Blimp-1 in the B-cell populations in all 11 MM patients but in none of 11 healthy subjects. We hypothesize that premature Blimp-1 expression coupled to altered and deficient Pax5 expression causes some proliferating B cells to prematurely differentiate to plasma cells in MM.