Identification and characterization of a novel monocyte subpopulation in human peripheral blood

With the aid of two-color immunofluorescence and flow cytometry, a new subset of cells coexpressing CD14 and CD16 antigens can be identified in human peripheral blood. Using the monoclonal antibody My4, these CD14+/CD16+ cells account for 2.2% of the mononuclear cells and form about 13% of all cells identified by the monocyte-specific CD14 monoclonal antibody. The CD14+/CD16+ cells can be assigned to the monocyte lineage based on typical morphology, on expression of additional monocyte-associated molecules, on the ability to form reactive oxygen intermediates and on the expression of monocyte-specific NaF-sensitive esterase. Light scatter analysis revealed lower forward angle and right angle light scatter for the CD14+/CD16+ cells compared with the regular monocytes, and the average cell size was determined to be 13.8 and 18.4 microns, respectively. Expression of class II antigens on these "small monocytes" was twofold higher compared with the regular monocytes. By contrast, the capacity to perform adherence to plastic surfaces, as well as the ability to phagocytize antibody-coated erythrocytes was clearly reduced in the CD14+/CD16+ monocyte subset as compared with the regular monocytes. Hence the CD14+/CD16+ cells appear to represent a new monocyte subset with a distinct functional repertoire. A survey of various tissues revealed that a large proportion of the alveolar macrophages, but not of the peritoneal macrophages, express the CD14+/CD16+ phenotype.     

Localization and characterization of a novel secreted protein SCUBE1 in human platelets

OBJECTIVE: The aim of the study was to investigate the protein expression and function of a novel secreted protein in the vascular system, named SCUBE1 for signal peptide, CUB (Complement proteins C1r/C1s, Uegf, and Bmp1) and epidermal growth factor-like (EGF)-like domain containing protein 1. METHODS AND RESULTS: Immunohistochemical analysis demonstrated that the SCUBE1 staining is mainly confined to the intravascular platelet-rich thrombus in vascular tissue samples. While quantitative real-time RT-PCR verified that the SCUBE1 mRNA is expressed in human platelets, numerous immunolocalization techniques revealed that the preformed SCUBE1 protein is stored in the alpha-granules and translocated to the surface upon platelet stimulation. A smaller SCUBE1 fragment, possibly formed by limited proteolysis after being released from the storage granules, was detected in thrombus lysate by Western blot analysis. Interestingly, deposition of SCUBE1 into the subendothelial matrix of the atherosclerotic plaques was evidenced by immunohistochemistry. In addition, studies of platelet adhesion and ristocetin-induced platelet agglutination showed that fragments containing the amino-terminal EGF-like repeats were able to support platelet adhesion and enhance the ristocetin-induced platelet agglutination, respectively. CONCLUSION: These data suggest that platelet-derived SCUBE1 could function as a novel adhesive molecule and its matrix-bound and soluble fragments may play critical (patho)physiological roles in cardiovascular biology.     

Lactocrine programming of female reproductive tract development: environmental connections to the reproductive continuum

For eutherian mammals a continuum of maternal support insures that development of progeny follows an optimal program. Beginning in utero, such support extends into the early neonatal period when bioactive factors are communicated from mother to offspring in colostrum/milk. Defined as lactocrine signaling, communication of milk-borne bioactive factors from mother to offspring as a consequence of nursing is important for development of somatic tissues, including the female reproductive tract (FRT). Data for the domestic pig indicate that lactocrine signaling contributes to the maternal continuum of factors that define the developmental program and determine the developmental trajectory of FRT tissues during early neonatal life. Both naturally occurring and manmade factors of environmental origin can be communicated to neonates in milk and affect development with lasting consequences. Here, evidence for lactocrine programming of FRT development and the potential for environmental endocrine disruption of this process are reviewed.     

Circulating immunity protects the female reproductive tract from Chlamydia infection

Anatomical positioning of memory lymphocytes within barrier tissues accelerates secondary immune responses and is thought to be essential for protection at mucosal surfaces. However, it remains unclear whether resident memory in the female reproductive tract (FRT) is required for Chlamydial immunity. Here, we describe efficient generation of tissue-resident memory CD4 T cells and memory lymphocyte clusters within the FRT after vaginal infection with Chlamydia. Despite robust establishment of localized memory lymphocytes within the FRT, naïve mice surgically joined to immune mice, or mice with only circulating immunity following intranasal immunization, were fully capable of resisting Chlamydia infection via the vaginal route. Blocking the rapid mobilization of circulating memory CD4 T cells to the FRT inhibited this protective response. These data demonstrate that secondary protection in the FRT can occur in the complete absence of tissue-resident immune cells. The ability to confer robust protection to barrier tissues via circulating immune memory provides an unexpected opportunity for vaccine development against infections of the FRT.

Naïve CD4 T cells circulate through lymphoid tissues and blood until an infection initiates lymphocyte clonal expansion and the acquisition of specific effector function (1, 2). Expanded effector CD4 T cells access inflamed or infected tissues where they regulate pathogen control and coordinate tissue repair (3, 4). In the aftermath of this host response, an elevated frequency of memory CD4 T cells returns to circulation, and a discrete population of noncirculating memory lymphocytes is retained within the tissues (5, 6). For many barrier tissues, the noncirculating tissue-resident memory T cells (T_RM) are critical for the rapid deployment of secondary immune responses upon pathogen reexposure (6–11). The female reproductive tract (FRT) is a barrier tissue that is thought to depend heavily on T_RM since it regularly encounters pathogens but lacks organized lymphoid tissues (12).The lack of organized lymphoid structures during steady-state and the immunologically restrictive nature of the FRT makes establishing immunity in this mucosal tissue a complex process. In certain contexts, lymphocytes as well as circulating antibody cannot easily enter the FRT mucosa (9, 10), suggesting that protective immune memory is contained within the tissue itself. Indeed, previous reports document the establishment of resident memory lymphocytes and mucosal antibody secretion as two important local protective mechanisms (7, 9, 13). Memory lymphocyte clusters (MLCs) are lymphoid structures that form at the interface of the FRT epithelium and lamina propria and consist of memory T cells and antigen presenting cells (13). Upon secondary infection, these MLCs can efficiently recruit effector cells in a CD4 T cell–dependent manner to clear FRT infections (9, 13). Thus, the generation of FRT MLCs is thought to be an important prerequisite for the development of new vaccines against important reproductive tract pathogens.A confusing aspect of this model is the observation that distal mucosal immunization in the lung can often induce local immunity in the FRT. It is not yet clear whether such distal immunization induces seeding the FRT with T_RM, formation of MLCs, and/or secretion of local antibody (14, 15). An alternative hypothesis is that distal immunization generates circulating memory responses that are recruited to the infected FRT and mediate pathogen clearance in the absence of T_RM or MLCs (15, 16).In this study, we examined whether local or distal immunization with the bacterial pathogen Chlamydia muridarum (Cm) is protective against intravaginal (I.Vag) challenge with Chlamydia. Our data reveal that CD4 T_RM efficiently populate the FRT and that MLCs are generated after local, but not distal, immunization. Using parabiosis, we demonstrate that the establishment of local resident memory lymphocytes in the FRT is dispensable for protective immunity. Indeed, circulating immunity induced locally or distally was completely sufficient for protection against local infection and FRT pathology. The depletion of lymphocytes revealed that local control of protection was mediated by circulating memory CD4 T cells.     

Neoplasia of the female reproductive tract: effects of hormone therapy

This review presents the data on the relationship between estrogen and estrogen plus progestin therapy in postmenopausal women and the occurrence of neoplasia in the endometrium, ovary, and uterine cervix. Estrogen only in women with an intact uterus consistently is shown to increase the incidence of endometrial cancer. Estrogen plus a cyclic or sequential progestin reduces the incidence of endometrial cancer to that found in never users. The duration of the progestin administration appears to be important with less than 10 d of progestin having an increased incidence of cancer after 5 yr of therapy. Continuous estrogen plus progestin does not increase the incidence of endometrial cancer. Estrogen and estrogen plus progestin effects on the occurrence of ovarian cancer are inconsistent. The data suggest a possible increase in ovarian epithelial tumors with >10 years use of estrogen only. There is no evidence of a change in the incidence of uterine cervical neoplasia with either estrogen or progestin.     

Identification and characterization of a novel isoform of hepatopoietin

AIM: To isolate a novel isoform of human HPO (HPO-205) from human fetal liver Marathon-ready cDNA and characterize its primary biological function. METHODS: 5'-RACE (rapid amplification of cDNA 5' ends) was used to isolate a novel isoform of hHPO in this paper. The constructed pcDNA(HPO-205), pcDNA(HPO) and pcDNA eukaryotic expression vectors were respectively transfected by lipofectamine method and the stimulation of DNA synthesis was observed by (3)H-TdR incorporation assay. Proteins extracted from different cells were analyzed by Western blot. RESULTS: A novel isoform of hHPO (HPO-205) encoding a 205 amino acid ORF corresponding to a translated production of 23 kDa was isolated and distinguished from the previous HPO that lacked the N-terminal 80 amino acids. The dose-dependent stimulation of DNA synthesis of HepG2 hepatoma cells by HPO-205 demonstrated its similar biological activity with HPO in vitro. The level of MAPK (Mitogen-activated protein kinase) phosphorylation by Western blot analysis revealed that HPO-205 might have the stronger activity of stimulating hepatic cell proliferation than that of HPO. CONCLUSION: A novel isoform of hHPO (HPO-205) was isolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure and function of hHPO, and provide the new way of thinking to deeply elucidate the biological roles of HPO/ALR.     

Identification and characterization of a novel expandable adult stem/progenitor cell population in the human exocrine pancreas

It is a general opinion that tissue-specific stem cells are present in adult tissues but their specific properties remain elusive. They are rare in tissues and heterogeneous; in addition, their identification and the characterization of their progeny has encountered technical difficulties. In particular, the existence of pancreatic stem cells remains elusive because specific markers for their identification are not available. We established a method for the isolation of a population of stem/progenitor cells from the human exocrine pancreas, and propose it as a model for other human compact organs. We also used markers that identified and finally characterized these cells. Spheroids with self-replicative potential were obtained from all specimens. The isolated population contained a subset of CD34+ CD45- cells and was able to generate, in appropriate conditions, colonies that produce insulin. We obtained evidence that most freshly isolated spheroids, when co-cultured with the c-kit positive neuroblastoma cell line LAN 5, produced a c-kit positive progeny of cells larger in their cytoplasmic content than the original spheroid population, with elongated morphology resembling the neuronal phenotype. We identified a novel predominant functional type of stem/progenitor cell within the human exocrine pancreas, able to generate insulin-producing cells and potentially non-pancreatic cells.     

Identification and characterization of Magmas, a novel mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction

OBJECTIVE: The aim of this study was to identify granulocyte-macrophage colony-stimulating factor (GM-CSF) responsive genes. MATERIALS AND METHODS: Potential GM-CSF responsive genes were identified by comparing the mRNA expression pattern of the murine myeloid cell line PGMD1 grown in either interleukin-3 (IL-3) or GM-CSF by differential display. Human and murine cDNA clones of one of the bands having increased expression in GM-CSF were isolated. mRNA expression of the gene was examined by Northern blot. Immunohistochemistry and studies with a green fluorescent fusion protein were used to determine its intracellular location. Growth factor-stimulated proliferation of PGMD1 cells transfected with constitutively expressed sense and anti-sense cDNA constructs of the gene was measured by 3H-thymidine incorporation. RESULTS: A gene, named Magmas (mitochondria-associated granulocyte macrophage CSF signaling molecule), was shown to be rapidly induced when cells were switched from IL-3 to GM-CSF. Analysis of the amino acid sequence of Magmas showed it contained a mitochondrial signal peptide, but not any other known functional domains. The human and murine clones encode nearly identical 13-kDa proteins that localized to the mitochondria. Magmas mRNA expression was observed in all tissues examined. PGMD1 cells that overexpressed Magmas proliferated similarly to untransfected cells when cultured in IL-3 or GM-CSF. In contrast, cells with reduced protein levels grew normally in IL-3, but had impaired proliferation in GM-CSF. CONCLUSION: Magmas is a mitochondrial protein involved in GM-CSF signal transduction.     

Giant cell arteritis of the female reproductive tract associated with temporal arteritis

A woman with giant cell arteritis of the reproductive tract is described. This is the 15th such case reported. This diagnosis led to the subsequent finding of biopsy proven temporal arteritis. The patient's illness responded favorably to corticosteroid therapy.     

Purification and partial characterization of a novel thyroxine-binding protein (27K protein) from human plasma

T4-binding globulin (TBG) prepared from human plasma by the standard three-step procedure (T4-agarose affinity chromatography, anion exchange chromatography, and gel filtration) often shows in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in addition to the expected 54K band, another with a mol wt of 27,000 (27K protein). The two proteins can be separated after the three-step procedure by chromatofocusing (because of different isoelectric points, 4.2-4.8 for TBG and 5.0-5.2 for 27K protein) or by T4-aragose chromatography eluting with a linear gradient of T4 (TBG is eluted between 10(-10) and 10(-9) M T4, 27K protein between 10(-8) and 10(-7) M T4). The 27K protein does not appear to be a fragment of TBG since 1) it does not displace [125I]TBG bound to anti-TBG monoclonal antibodies; and 2) absorption of polyclonal antibody reacting with both TBG and 27K protein with sera from TBG-deficient patients completely prevents [125I]27K protein binding, while only slightly affecting [125I]TBG binding. On the other hand, 27K protein is not simply a contaminant devoid of biological activity, but is a T4-binding protein, as supported by the following findings: 1) it covalently binds [125I]T4 by photoaffinity labeling, and this binding can be almost completely prevented by excess T4; 2) equilibrium dialysis shows two equivalent T4-binding sites per 66K, with an association constant of 0.85 X 10(7) M-1, intermediate between albumin and prealbumin; and 3) tryptophanyl fluorescence analysis shows quenching of 37% of the fluorescence when the protein is titrated with T4. The 27K protein appears as a single 27K band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pH 8.8, but under nondenaturing nonreducing conditions mostly remains at the origin of the gel; a fraction enters the gel and migrates slightly ahead of albumin. This electrophoretic pattern is distinct from those of albumin, prealbumin, and TBG. In immunoelectrophoresis in agar at pH 8.6, 27K protein moves slightly faster than TBG. The results of equilibrium sedimentation indicate a mol wt of 66,000, suggesting that the 27K protein might exist as a dimer. These data indicate that the 27K protein is a previously unrecognized T4-binding protein with a low affinity for the hormone. Further studies are required to clarify its physiological role in the transport of circulating thyroid hormones.