Characterization of host CD4+ T lymphocytes in mice neonatally tolerized to alloantigens

BALB/c mice injected at birth with semi-allogeneic F1 spleen cells become tolerant to alloantigens as shown by their CTL unresponsiveness to the corresponding alloantigen and the persistence of donor F1 cells into the BALB/c host. Moreover, these mice develop a transient systemic lupus erythematosis-like autoimmune syndrome characterized by splenomegaly, glomerulonephritis, thrombocytopenia and abnormal serological findings, such as several autoantibodies and IgG1 hypergammaglobulinemia. Recent studies done in our laboratory have shown that donor F1 B cells persisting in the host are responsible for the production of autoantibodies and must be activated in vivo by the host CD4⁺ T lymphocytes in a MHC class II-restricted fashion. In the present work, we have focused our attention on the ability of splenic CD4⁺ T cells recovered at different periods from BALB/c mice injected at birth with (CBA/Ca × BALB. Igh^b) Fl spleen cells to interact with and activate F1 semi-allogeneic spleen cells in vitro. We show that (i) only CD4⁺ T cells from 2- and 3-week-old tolerant BALB/c mice preferentially produce IL-4 and IL-5 in response to a F1 semi-allogeneic in vitro stimulation, (ii) CD4⁺ T cells purified from 3-week-old tolerant BALB/c mice are able to induce in vitro IgG and IgM production by F1 B cells. Taken together, these results strongly suggest that host CD4+ T cells, belonging to the TH2 subset progressively lose their reactivity towards the F1 semi-allogeneic persistent B cells, reaching a state of unresponsiveness that correlates with the disappearance of serum autoantibodies and autoimmune pathology.     

CD4-independent protective cytotoxic T cells induced in early life by a non-replicative delivery system based on virus-like particles

The relative immaturity of the neonatal immune system limits CD4(+) Th1 and cytotoxic T lymphocyte (CTL) responses, and represents a significant challenge for the development of vaccines against intracellular pathogens. In this report, we demonstrate the ability of a non-replicative delivery system based on parvovirus-like particles (VLP) to induce CTL responses in the neonatal period. A single immunization of 1-week-old BALB/c mice with recombinant VLP carrying a CD8(+) T cell determinant from lymphocytic choriomeningitis virus (VLP-LCMV) induced antigen-specific CD8(+) cytotoxic T cells that were similar to those elicited by adult immunization, as assessed by cytotoxic activity, interferon (IFN)-gamma secretion, cytotoxic precursor cell frequencies, in vitro avidity for antigen and protective activity against viral challenge. These CTL responses are elicited within 2 weeks of a single immunization, in the absence of adjuvant and independently of the presence and help of CD4(+) T cells, highlighting the potential of VLP as candidate vaccine vectors in early life.     

What is the meaning of colorectal transit time measurement?

This study was done to understand the different available methods used to calculate colorectal transit times. A single abdominal radiograph is taken following six successive daily ingestions of the same number of identical radiopaque markers. This method correlates well (P < 0.001) with that using a single ingestion of markers with daily x-ray films until total expulsion. In techniques used to measure colorectal transit time with multiple ingestion of markers, the number of days of ingestion depends on the kinetics of marker defecation. This was found to differ markedly in various groups of control subjects and constipated patients (P < 0.001) and can be used to obtain reliable data, even in subjects with severe constipation. When they ingest 20 markers, constipated patients are found to retain eight or more markers three days after ingestion, and taking a plain film of the abdomen on that day is sufficient to make a diagnosis of constipation. Transit time studies are reproducible from month to month in patients with an irritable bowel syndrome. Control subjects who claim that their bowel habits are not modified by stress have shorter transit times, similar in both sexes, than those who say they are (P <0.001). This may explain why a large percentage of constipated patients have been found by most authors to have normal colorectal transit times. The choice of control subjects is thus a key element in studies of functional bowel motor disorders. Stool frequency and consistency, in health, correlate only to rectosigmoid transit time.     

Molecular Identity of Hematopoietic Precursor Cells Emerging in the Human Embryo

It is now accepted from studies in animal models that hematopoieticstem cells emerge in the para-aortic mesoderm-derived aorta-gonad-mesonephros region of the vertebrate embryo. We have previously identified the equivalent primitive hematogenous territory in the 4- to 6-week human embryo, under the form ofCD34⁺CD45⁺Lin highproliferative potential hematopoietic cells clustered on the ventralendothelium of the aorta. To characterize molecules involved in initialstem cell emergence, we first investigated the expression in thatterritory of known early hematopoietic regulators. We herein show thataorta-associated CD34⁺ cells coexpress the tal-1/SCL,c-myb, GATA-2, GATA-3, c-kit, and flk-1/KDR genes, as do embryonic andfetal hematopoietic progenitors later present in the liver and bonemarrow. Next, CD34⁺CD45⁺ aorta-associatedcells were sorted by flow cytometry from a 5-week embryo and a cDNAlibrary was constructed therefrom. Differential screening of thatlibrary with total cDNA probes obtained from CD34⁺embryonic liver cells allowed the isolation of a kinase-related sequence previously identified in KG-1 cells. In addition to emerging blood stem cells, KG-1 kinase is also strikingly expressed in alldeveloping endothelial cells in the yolk sac and embryo, which suggestsits involvement in the genesis of both hematopoietic and vascular celllineages in humans.     

Measurement of gastric emptying in dyspeptic patients: effect of a new gastrokinetic agent (cisapride).

Symptoms suggesting gastroparesis in patients without gastric outlet obstruction are very common but their relation to an objective delay of gastric emptying has been poorly investigated. A dual isotopic technique was used to evaluate patients with non-obstructive dyspepsia (idiopathic and secondary) (part 1) and to assess the effects of a new gastrokinetic agent: cisapride, on gastric emptying in such patients (part 2). Sixty patients with postprandial dyspeptic symptoms (vomiting, nausea, gastric bloating or full feeling) and without lesions at upper endoscopy were studied. They were distributed into three groups: idiopathic dyspepsia (n = 31), postvagotomy dyspepsia (n = 16) and dyspepsia secondary to medical disorders (n = 13). All patients ingested the same ordinary meal; 99mTc sulphur colloid tagged egg white was the solid phase marker and 111In chloride was the liquid phase marker. In part 1, evaluation of gastric emptying in the first 50 patients shows a delay of gastric emptying rate of solids and liquids as compared with controls. Striking differences separate the three groups of patients, however, percentages of delayed gastric emptying rate of solids and or liquids averaged 90% in postvagotomy or secondary dyspepsia groups whereas it was 44% in idiopathic dyspepsia group. Moreover, liquid emptying rate was often the only one impaired in idiopathic dyspepsia, and in 12 of the 27 patients of this group the faster emptying rate of liquids as compared with that of solids (always found in normal subjects), could not be evidenced. In part 2, 10 patients entered a double blind cross over study of cisapride (8 mg intravenously). A significant increase of solid (p<0.01) and liquid (p<0.05) emptying rates was found in patients with initial gastric emptying delay. This study emphasises the importance of an objective evaluation of gastric emptying in the presence of symptoms of gastric stasis and suggests that specific local acting therapy may be useful in patients with identified abnormal gastric emptying.     

Heparan sulfate mimetics can efficiently mobilize long-term hematopoietic stem cells

Background

Although mobilization of hematopoietic stem cells and hematopoietic progenitor cells can be achieved with a combination of granulocyte colony-stimulating factor and plerixafor (AMD3100), improving approaches for hematopoietic progenitor cell mobilization is clinically important.

Design and Methods

Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the extracellular matrix that regulates biology of hematopoietic stem cells. We studied the effects of a new family of synthetic oligosaccharides mimicking heparan sulfate on hematopoietic stem cell mobilization. These oligosaccharides were administered intravenously alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 in mice. Mobilized hematopoietic cells were counted and phenotyped at different times and the ability of mobilized hematopoietic stem cells to reconstitute long-term hematopoiesis was determined by competitive transplantation into syngenic lethally irradiated mice followed by secondary transplantation.

Results

Mimetics of heparan sulfate induced rapid mobilization of B-lymphocytes, T-lymphocytes, hematopoietic stem cells and hematopoietic progenitor cells. They increased the mobilization of hematopoietic stem cells and hematopoietic progenitor cells more than 3-fold when added to the granulocyte colony-stimulating factor/AMD3100 association. Hematopoietic stem cells mobilized by mimetics of heparan sulfate or by the granulocyte colony-stimulating factor/AMD3100/mimetics association were as effective as hematopoietic stem cells mobilized by the granulocyte colony-stimulating factor/AMD3100 association for primary and secondary hematopoietic reconstitution of lethally irradiated mice.

Conclusions

This new family of mobilizing agents could alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 mobilize a high number of hematopoietic stem cells that were able to maintain long-term hematopoiesis. These results strengthen the role of heparan sulfates in the retention of hematopoietic stem cells in bone marrow and support the use of small glyco-drugs based on heparan sulfate in combination with granulocyte colony-stimulating factor and AMD3100 to improve high stem cell mobilization, particularly in a prospect of use in human therapeutics.