Pre-implantation development of cattle embryos produced from fresh bull semen enriched for X- chromosome-bearing spermatozoa using a monoclonal antibody.

Immunological approaches are gaining attention as a convenient and economical method for sex-sorting mammalian spermatozoa. A monoclonal antibody (WholeMom™) has previously been reported to cause agglutination of Y-chromosome-bearing spermatozoa in frozen-thawed semen for gender preselection. However, its usefulness for gender preselection in fresh semen and subsequent in vitro fertilization (IVF) after freeze-thawing has not been reported. This study investigated the in vitro development of cattle embryos produced from fresh bull semen pre-treated with WholeMom™ monoclonal antibody. Results showed that antibody-treated, non-agglutinated spermatozoa (presumably X-chromosome-bearing spermatozoa) could fertilize cattle oocytes in vitro. However, embryos generated from non-agglutinated (enriched in X-chromosome-bearing spermatozoa) had a lower (p < 0.05) ability to cleave (66.4 ± 2.5% vs. 75.1 ± 3.3%) than those of non-treated control sperm. Nevertheless, the percentage of blastocysts developed from cleaved embryos did not differ (p > 0.05) between the groups (34.8 ± 3.7% vs. 35.8 ± 3.4%). Duplex PCR of blastocysts, using a bovine-specific universal primer pair and a Y-chromosome-specific primer pair, showed a sex ratio of 95.8% females from sex-sorted spermatozoa, which was higher than those of non-treated control spermatozoa (46.4%). In conclusion, the results of the present study suggest that monoclonal antibody-based enrichment of X- chromosome-bearing spermatozoa can be applied to fresh bull semen without compromising their post-fertilization early embryonic development to the blastocyst stage. Future studies should investigate the term development and sex ratio of calves from antibody-treated spermatozoa.

5-Azacytidine improves the meiotic maturation and subsequent in vitro development of pig oocytes.

Treatment of donor cells and/or cloned embryos with cytidine analogues, having an Aza group at its 5th carbon (5-Aza), such as 5-Azacytidine (5-Aza-C) or 5-Aza-2'-deoxycytidine (5-Aza-dC) improves the in vitro development of cloned embryos produced by somatic cell nuclear transfer (SCNT). In vitro maturation (IVM) of immature pig oocytes treated with 5-Aza-C not only results in greater (P < 0.05) meiotic maturation to the MII stage but also enhances the capacity of 5-Aza-C treated oocytes for early embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF) or SCNT in a dose-dependent manner (0-10 µM). Cloned embryos generated from 5-Aza-C (0.01 µM) treated oocytes had an increased capacity to develop to the blastocyst stage (14.1 ±â€¯1.5% compared with 9.6 ±â€¯1.8%), greater probability of hatching (61.8 ±â€¯1.5% compared with 45.0 ±â€¯3.9%) and contained a greater number of cells per blastocyst (38.5 ±â€¯4.4 compared with 30.5 ±â€¯3.4) than those produced from non-treated control oocytes (P < 0.05). Data from the present study indicate that treatment of oocytes with 5-Aza-C may be an important approach to enhance the meiotic maturation and subsequent in vitro development of pig embryos. Future studies should be conducted to determine the underlying mechanism of improved early embryonic development of 5-Aza-C treated oocytes.

A New Triggering Receptor Expressed on Myeloid Cells (TREM) Family Member, TLT-6, is Involved in Activation and Proliferation of Macrophages.

The triggering receptor expressed on myeloid cells (TREM) family, which is abundantly expressed in myeloid lineage cells, plays a pivotal role in innate and adaptive immune response. In this study, we aimed to identify a novel receptor expressed on hematopoietic stem cells (HSCs) by using in silico bioinformatics and to characterize the identified receptor. We thus found the TREM-like transcript (TLT)-6, a new member of TREM family. TLT-6 has a single immunoglobulin domain in the extracellular region and a long cytoplasmic region containing 2 immunoreceptor tyrosine-based inhibitory motif-like domains. TLT-6 transcript was expressed in HSCs, monocytes and macrophages. TLT-6 protein was up-regulated on the surface of bone marrow-derived and peritoneal macrophages by lipopolysaccharide stimulation. TLT-6 exerted anti-proliferative effects in macrophages. Our results demonstrate that TLT-6 may regulate the activation and proliferation of macrophages.

Effect of feeding fermented rice punch on oestrous induction in anoestrous bitches.

INTRODUCTION: As a consequence of poor productivity caused by a long anoestrous period, considerable research effort has been given to oestrus induction in dogs to enhance the productivity of young dogs and to preserve breeds. MATERIALS AND METHODS: Oestrus was induced in 30 anoestrous bitches more than three months after the last oestrus. Bitches orally received fermented rice punch with or without bromocriptine once daily for 21 consecutive days. The bitches were divided into two groups (n=10 per group): Group (1) fed fermented rice punch and Group (2) administered bromocriptine (100 µg/kg/day) and fed fermented rice punch. RESULTS: The concentration of dopamine in fermented rice punch was 47.2 mg/kg (parts per million). Six of 10 (60.0 per cent) and seven of 10 (70.0 per cent) bitches showed pro-oestrual bleeding in Groups 1 and 2, respectively. The mean and median values (min-max) to oestrus induction was not significantly different between Groups 1 and 2 (9.7±7.3, 6.5 (3-22) and 11.3±6.6, 7.9 (5-21) days) after treatment commencement (P>0.05). The pregnancy rate was very similar between Groups 1, 2 (66.0%) and control (66.0, 57.0 and 50.0 per cent). The mean and median values (min-max) of pups per bitch are also not significantly different between Groups 1, 2 and control (7.0±1.8, 7.0 (5-9) and 7.5±2.1, 7.5 (5-10) and 7.0±0, 7.0 (7-7)). CONCLUSION: We suggest that rice punch effectively induces oestrus in bitches.

Monoclonal antibody against α-actinin 4 from human umbilical vein endothelial cells inhibits endothelium-dependent vasorelaxation.

BACKGROUND: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. METHODS: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. RESULTS: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. CONCLUSIONS: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.

Mesenchymal stem cells regulate the proliferation of T cells via the growth-related oncogene/CXC chemokine receptor, CXCR2.

Mesenchymal stem cells (MSCs) have known to induce immunosuppressive properties by preventing T cell proliferation. However, it is remains unclear how MSCs inhibit T cell proliferation. To identify the factor that inhibits T cell proliferation, we conducted a cytokine array analysis of culture medium from a co-culture of MSCs and T cells and found that the chemokines, CXCL1, 2 and 3, were induced in T cells. MSCs also induced the expression of the CXCR2 receptor on T cell surface. Particularly, CXCL3 inhibited proliferation and increased apoptosis in T cells, which were reversed by CXCR2 inhibitor treatment. Moreover, CXCL3 decreased JAK2, STAT3, and AKT phosphorylation and these responses were also abolished by CXCR2 inhibitor treatment. MSCs suppressed the proliferation of T cells into tumor tissue. Collectively, these data demonstrate that MSCs directly regulate T cell proliferation by induction of CXCL3 chemokine and its receptor, CXCR2 on the surface in T cells.

Increased HoxB4 Inhibits Apoptotic Cell Death in Pro-B Cells.

HoxB4, a homeodomain-containing transcription factor, is involved in the expansion of hematopoietic stem cells and progenitor cells in vivo and in vitro, and plays a key role in regulating the balance between hematopoietic stem cell renewal and cell differentiation. However, the biological activity of HoxB4 in other cells has not been reported. In this study, we investigated the effect of overexpressed HoxB4 on cell survival under various conditions that induce death, using the Ba/F3 cell line. Analysis of phenotypical characteristics showed that HoxB4 overexpression in Ba/F3 cells reduced cell size, death, and proliferation rate. Moreover, the progression from early to late apoptotic stages was inhibited in Ba/F3 cells subjected to HoxB4 overexpression under removal of interleukin-3-mediated signal, leading to the induction of cell cycle arrest at the G2/M phase and attenuated cell death by Fas protein stimulation in vitro. Furthermore, apoptotic cell death induced by doxorubicin-treated G2/M phase cell-cycle arrest also decreased with HoxB4 overexpression in Ba/F3 cells. From these data, we suggest that HoxB4 may play an important role in the regulation of pro-B cell survival under various apoptotic death environments.

Alpha 1,3-galactosyltransferase deficiency in pigs increases sialyltransferase activities that potentially raise non-gal xenoantigenicity.

We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level of α-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH), which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production of N-glycolylneuraminic acid (Neu5Gc) due to an increase of α2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.

T lymphocytes derived from human cord blood provide effective antitumor immunotherapy against a human tumor.

BACKGROUND: Although the graft-versus-tumor (GVT) effect of donor-derived T cells after allogeneic hematopoietic stem cell transplantation has been used as an effective adoptive immunotherapy, the antitumor effects of cord blood (CB) transplantation have not been well studied. METHODS: We established the animal model by transplantation of CB mononuclear cells and/or tumor cells into NOD/SCID mice. The presence of CB derived T cells in NOD/SCID mice or tumor tissues were determined by flow cytometric and immunohistochemical analysis. The anti-tumor effects of CB derived T cells against tumor was determined by tumor size and weight, and by the cytotoxicity assay and ELISPOT assay of T cells. RESULTS: We found dramatic tumor remission following transfer of CB mononuclear cells into NOD/SCID mice with human cervical tumors with a high infiltration of CD3+ T cells in tumors. NOD/SCID mice that receive neonatal CB transplants have reconstituted T cells with significant antitumor effects against human cervical and lung tumors, with a high infiltration of CD3+ T cells showing dramatic induction of apoptotic cell death. We also confirmed that T cells showed tumor specific antigen cytotoxicity in vitro. In adoptive transfer of CD3+ T cells into mice with pre-established tumors, we observed much higher antitumor effects of HPV-specific T cells by ELISPOT assays. CONCLUSIONS: Our results show that CB derived T lymphocytes will be useful for novel immunotherapeutic candidate cells for therapy of several tumors in clinic.

High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells.

OBJECTIVE: To develop an efficient freezing method suitable for large-scale cryopreservation of human embryonic stem cells (hESCs). DESIGN: Experimental study. SETTING: Research institute. PATIENT(S): None. INTERVENTION(S): Two genetically modified hESC lines, H9-EF1-GFP and CHA-hES3-EF1-GFP, were cryopreserved in cryovials using a combination of two equilibration methods (one-step and stepwise) and two cooling vehicles (cryo-container and program-controlled freezer). After thawing, the survival and differentiation rate were compared among groups. MAIN OUTCOME MEASURE(S): The hESC survival was assessed by alkaline phosphatase staining and differentiation status was determined by flow cytometry using an SSEA-4 antibody. RESULT(S): In both H9-EF1-GFP and CHA-hES3-EF1-GFP cells, the survival rate was highest in the group using stepwise equilibration and program-controlled freezer, and lowest in the group using one-step equilibration and cryo-container. In the groups using cryo-container, the survival and the frequency of undifferentiated cells in both cell lines was highly improved in a stepwise equilibration compared with one-step. Thawed hESCs were positively stained with pluripotent markers SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The karyotypes and expression of three germ layer markers in both cell lines were not changed after freezing/thawing. CONCLUSION(S): The stepwise equilibration of Knockout Serum Replacement and cryoprotectant during freezing and thawing resulted in higher survival rates by reducing osmotic damage irrespective of cooling vehicles.

Doxorubicin exerts cytotoxic effects through cell cycle arrest and Fas-mediated cell death.

Doxorubicin (DOX) is involved in the induction of DNA damage, inhibition of cell proliferation, impairment of mitochondria, and cell death. To determine the biological effects of DOX in murine lymphocytes, we analyzed cell proliferation, cell cycle status, and apoptosis in Ba/F3 and EL4 lymphoid cells. DOX treatment resulted in significant cellular morphological alteration with increased intracellular granularity and cell size. DOX inhibited cell proliferation through cell cycle arrest at the G(2)/M phase as well as by cell death. In addition, DOX treatment dramatically upregulated Fas expression and enhanced caspase activation to promote intracellular apoptotic signaling for cell death. Treatment with an agonistic antibody stimulated Fas and accelerated the cell death effects. In conclusion, we demonstrate that DOX induces cell cycle arrest and apoptosis by increased Fas expression and ultimately results in enhanced cell death.

CD137 ligand-mediated reverse signals increase cell viability and cytokine expression in murine myeloid cells: involvement of mTOR/p70S6 kinase and Akt.

Cross-linking of CD137 ligand (CD137L), a member of the TNF family, with recombinant CD137-Fc (rCD137-Fc) protein enhanced adherence of bone marrow-derived macrophages, and increased the expression of ICAM-1, IL-1beta, IL-6, M-CSF and phosphotyrosine proteins. In RAW264.7 cells, a murine myeloid cell line, rCD137-Fc not only increased adherence but also cell multiplication, in a manner comparable to LPS or M-CSF. In addition, it up-regulated expression of IL-1beta, IL-1 receptor antagonist, IL-6, COX2, tenascin C, neuropeptide Y and M-CSF mRNA. Neutralization of M-CSF by incubating the RAW264.7 cells with anti-M-CSF mAb did not prevent the CD137L signal-induced viability. Viability was blocked by PP2, an Src tyrosine kinase inhibitor, rapamycin, an mTOR inhibitor and LY294002, a PI3K inhibitor, but not by Wortmannin, another PI3K inhibitor. Cross-linking of CD137L increased phosphorylation of Akt and p70S6 kinase. The latter was blocked by PP2, rapamycin or LY294002, but not by Wortmannin, whereas phosphorylation of Akt was blocked by LY294002 or Wortmannin. These findings demonstrate that reverse signals evoked by CD137L regulate immune functions in macrophages.

Implication of human OCT4 transactivation domains for self-regulatory transcription.

OCT4 plays a crucial role in pluripotency and self-renewal of embryonic stem cells. OCT4 is also expressed in testicular germ cell tumors (GCTs), suggesting the important function of OCT4 as an oncogenic factor in GCTs. To understand the molecular mechanism of human OCT4 (hOCT4) in tumorigenesis as well as stemness, we identified hOCT4 transactivation domains in human embryonic carcinoma cells. Context analyses of heterologous GAL4 and natural hOCT4 revealed that each N-terminal domain or C-terminal domain independently stimulated transcriptional activity, and that both domains are required for synergistic transactivation by deletion mapping analysis. Dose-dependent overexpression of exogenous hOCT4 significantly decreased the transcriptional activity of the hOCT4 promoter. This inhibition was reversed by the removal of one or both domains. These results suggest that the inhibitory effect of hOCT4 is mediated by transactivation domains, and that the self-regulation of hOCT4 may be mediated via a negative feedback loop in pluripotent cells.

Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells.

Genetic modification of hematopoietic stem cells holds great promise in the treatment of hematopoietic disorders. However, clinical application of gene delivery has been limited, in part, by low gene transfer efficiency. To overcome this problem, we investigated the effect of retronectin (RN) on lentiviral-mediated gene delivery into hematopoietic progenitor cells (HPCs) derived from bone marrow both in vitro and in vivo. RN has been shown to enhance transduction by promoting colocalization of lentivirus and target cells. We found that RN enhanced lentiviral transfer of the VENUS transgene into cultured c-Kit(+) Lin(-) HPCs. As a complementary approach, in vivo gene delivery was performed by subjecting mice to intra-bone marrow injection of lentivirus or a mixture of RN and lentivirus. We found that co-injection with RN increased the number of VENUS-expressing c-Kit(+) Lin(-) HPCs in bone marrow by 2-fold. Further analysis of VENUS expression in colony-forming cells from the bone marrow of these animals revealed that RN increased gene delivery among these cells by 4-fold. In conclusion, RN is effective in enhancing lentivirus-mediated gene delivery into HPCs.

Two potent transactivation domains in the C-terminal region of human NANOG mediate transcriptional activation in human embryonic carcinoma cells.

The core embryonic stem cell transcription factors Oct4, Sox2, and Nanog are expressed in germ cell tumors (GCTs) and have been proposed to play a regulatory role in tumorigenesis. However, little is known about the mechanism of regulation of tumorigenesis by the complicated network of these proteins. Nanog is a novel homeobox-containing transcription factor that is expressed in pluripotent cells as well as GCTs. To understand the molecular and functional role of human NANOG (hNANOG) in germ cells, mutagenesis of the C-terminal domain (CD) of hNANOG and transient transfection assays in NCCIT human embryonic carcinoma cells were carried out to identify critical transactivation motifs. We divided the CD into three putative functional subdomains, CD1, tryptophan-repeat (WR) subdomain, and CD2. WR subdomain and CD2 independently contained transcriptional potential and, in combination, had a synergistic effect on transcriptional activity, while CD1 was transcriptionally inactive. The glutamine (Q) motif in WR subdomain, and multiple acidic residues in CD2 were required for maximal and synergistic transcriptional activation by the hNANOG CD. The results of the current study contribute to a better understanding of the complicated molecular machinery of stem cell transcription factors and their role in unregulated proliferation in germ cell tumorigenesis.

Characterization of a stem cell population in lung cancer A549 cells.

We isolated a stem cell subpopulation from human lung cancer A549 cells using FACS/Hoechst 33342. This side population (SP), which comprised 24% of the total cell population, totally disappeared after treatment with the selective ABCG 2 inhibitor fumitremorgin C. In a repopulation study, isolated SP and non-SP cells were each able to generate a heterogeneous population of SP and non-SP cells, but this repopulation occurred more rapidly in SP cells than non-SP. An MTT assay and cell cycle distribution analysis reveal a similar profile between SP and non-SP groups. However, in the presence of doxorubicin (DOX) and methotrexate (MTX), SP cells showed significantly lower Annexin V staining when compared to non-SP cells. Taken together, these results demonstrate that SP cells have an active regeneration capacity and high anti-apoptotic activity compared with non-SP cells. Furthermore, our GeneChip data revealed a heightened mRNA expression of ABCG2 and ABCC2 in SP cells. Overall these data explain why the SP of A549 has a unique ability to resist DOX and MTX treatments. Therefore, we suggest that the expression of the ABCG2 transporter plays an important role in the multidrug resistance phenotype of A549 SP cells.

Interaction of glycopolymers with human hematopoietic cells from cord blood and peripheral blood.

Polystyrene derivatives, poly[N-pvinylbenzyl-O-D-glucopyranosyl-(1-4)-D-glucoamide] (PV Maltose) and poly[N-p-vinylbenzyl-O-mannopyranosyl-(1-4)-D-glucoamide] (PV Mannose), which contain glucose and mannose moieties, respectively, have the specific binding ability with murine hematopoietic cells. In this study, we confirm the ability of these glycopolymers to interact specifically with human hematopoietic stem cells (HSCs) and mature cells derived from human cord blood (CB) and peripheral blood (PB). Using fluorescence isothiocyanate (FITC)-labeled glycopolymers, we observed that 98% to 93% of hematopoietic cells interacted very strongly with PV Mannose, and 63% of CB and 29% PB interacted with PV Maltose. Both glycopolymers bound better to cells from CB than from PB. Cytotoxic studies revealed that a 0.1 mM dose of PV Mannose induced apoptosis in 20% CB cells, in contrast to 3-5% PB cells. Furthermore, we demonstrated that all of CD34(+) HSCs of both origins bound specifically to PV Mannose, whereas 33-47% bound to PV Maltose. In addition, the majority of B cells (CD19(+)), T cells (CD3(+)), monocytes (CD14(+)), and erythrocytes (CD235a(+)) bound to PV Mannose, but a lower percentage interacted with PV Maltose. In vivo study, bone marrow, spleen, and liver tissues in NOD-SCID mice injected with PV Mannose conjugated CB, were detected PV Mannose positive hematopoietic cells. These data suggest that the use of PV Mannose and PV Maltose might be used for gene and drug delivery for hematopoietic cells and thus, may be useful in therapeutic settings.

Gene expression profiling of cancer stem cell in human lung adenocarcinoma A549 cells.

BACKGROUND: The studies on cancer-stem-cells (CSCs) have attracted so much attention in recent years as possible therapeutic implications. This study was carried out to investigate the gene expression profile of CSCs in human lung adenocarcinoma A549 cells. RESULTS: We isolated CSCs from A549 cell line of which side population (SP) phenotype revealed several stem cell properties. After staining the cell line with Hoechst 33342 dye, the SP and non-side population (non-SP) cells were sorted using flow cytometric analysis. The mRNA expression profiles were measured using an Affymetrix GeneChip(R) oligonucleotide array. Among the sixty one differentially expressed genes, the twelve genes inclusive three poor prognostic genes; Aldo-keto reductase family 1, member C1/C2 (AKR1C1/C2), Transmembrane 4 L six family member 1 nuclear receptor (TM4SF1), and Nuclear receptor subfamily 0, group B, member 1 (NR0B1) were significantly up-regulated in SP compared to non-SP cells. CONCLUSION: This is the first report indicating the differences of gene expression pattern between SP and non-SP cells in A549 cells. We suggest that the up-regulations of the genes AKR1C1/C2, TM4SF1 and NR0B1 in SP of human adenocarcinoma A549 cells could be a target of poor prognosis in anti-cancer therapy.

Extracellular ATP is involved in the induction of apoptosis in murine hematopoietic cells.

Extracellular nucleotides have multiple biological actions in processes such as proliferation, differentiation, chemotaxis, and cytokine secretion through P2X receptors on the cell surface. To determine the biological activity of adenosine triphosphate (ATP) and the expression of P2 nucleotide receptors in murine bone marrow-derived hematopoietic cells and stem cells/progenitor cells, we investigated the effects of ATP in assays of cell proliferation and cell death in vitro. Our results demonstrated that several subtypes of P2X receptors were expressed on hematopoietic cells and that P2X7, in particular, was partially expressed in hematopoietic stem cells/progenitor cells. In addition, stimulation of hematopoietic cells with high concentrations of ATP caused severe inhibition of cell proliferation despite the presence of cytokine stimulation. We analyzed the apoptotic effects of stimulation with several different dosages of ATP and confirmed the enhanced apoptotic activity in hematopoietic cells and progenitor cells. Antagonists, against P2X receptors and ATP, suramin and oxidized ATP, inhibited the induction of cell death for murine hematopoietic cells. Our data suggest that extracellular nucleotides may provide a novel and powerful tool for regulating the cell fate of hematopoietic stem cells.

A human mutant CD4 molecule resistant to HIV-1 binding restores helper T-lymphocyte functions in murine CD4-deficient mice.

CD4 is a cell surface glycoprotein that acts as a co-receptor for the T cell antigen receptor by binding to a non-polymorphic portion of MHC molecules. CD4 also functions as a receptor for human immunodeficiency virus type-I (HIV-1) because the viral envelope glycoprotein gp120 binds to CD4 with a high affinity. We have previously demonstrated that introduction of mutations into CD4 abolished the binding of gp120 and prevented HIV-1 from entering cells and spreading. However, whether introduction of such mutations into CD4 causes decreased binding to MHC and loss of function is yet to be determined. We generated transgenic mouse lines by injecting a mutant human CD4 (muthCD4) gene under a murine CD4 enhancer/promoter to ensure tissue and stage specific expression. To exclude the influence of endogenous murine CD4, transgenic mice were crossed with murine CD4-targeted mice to produce muthCD4 transgenic mice lacking endogenous CD4 (muthCD4TG/KO mice). In these mice, T lymphocytes expressing muthCD4 expanded and matured in the thymus and were present in the spleen and lymph nodes. They also activated B cells to mount an antibody response to a T-dependent antigen. The results from this study suggest that a human variant of CD4 modified to be resistant to HIV-1 binding can rescue the signaling for T cell development in the thymus in vivo, having helper T cell functions. Thus, further characterization of muthCD4 molecules should open the way to new HIV treatment modalities.