Search PubMed⌕ Search

Biomedical subjects

J R Klein

Publications and source records attributed to J R Klein.

At least 55 records · Page 3Linked to original sources
PubMed1996

Long-term outcome after fetal cardiac bypass: fetal survival to full term and organ abnormalities.

BACKGROUND: Earlier work suggests that fetal cardiac bypass is technically feasible but results in significant placental dysfunction. Many of the stimuli that initiate this placental dysfunction have been identified in the past several years and these involve fetal stress, extracorporeal surfaces, priming substances (maternal blood), and flow characteristics. Fetal survival with conventional methods of bypass has been far less than optimal. A novel fetal bypass circuit requiring no priming volume was designed incorporating an in-line axial flow pump (Hemopump, Johnson & Johnson Interventional Systems, Rancho Cordova, Calif.) and was demonstrated to have a marked beneficial effect on placental function. OBJECTIVE: The purpose of this study was to investigate the effect of this newly developed customized fetal bypass circuit on fetal survival and developing fetal organs. METHODS AND RESULTS: Nine fetuses at 122 to 126 days of gestation were subjected to fetal cardiac bypass via a transsternal approach, with a 16F single right atrial venous cannula and a 12F arterial cannula. Normothermic cardiac bypass was continued for 30 minutes at flow rates of 320 +/- 32 ml/kg. Of the nine fetuses, one fetus was stillborn 4 days after bypass and eight (89%) were delivered alive after progressing to term gestation. One lamb died of blunt trauma 1 day after birth. All other lambs (n = 7) thrived normally, and at 1 week of age they were subjected to autopsy. No gross hemorrhagic or thromboembolic lesions were detected in the organs examined including the brain. Microscopic examination of representative sections from all organs revealed mild pleural reaction in two lambs, and in two other lambs the hepatocytes showed evidence of mild increase in glycogen content, the significance of which is unknown in relation to fetal bypass. In one fetus that was aborted there was evidence of mild to moderate neuronal loss in the cerebral cortex. CONCLUSION: This study demonstrates that with improvements in fetal extracorporeal circuitry and techniques very favorable fetal outcome can be achieved. Further studies are necessary to evaluate the effects of bypass on fetal brain in an appropriate animal model. Advances in extracorporeal circuitry to suit the unique fetal physiology increase the possibility of future clinical application.

Animals↗
PubMed1996

Hemodynamic effects of epinephrine, bicarbonate and calcium in the early postnatal period in a lamb model of single-ventricle physiology created in utero.

OBJECTIVES: A reproducible fetal animal model of single-ventricle physiology was created to examine the effects of pharmacologic agents commonly used in the perinatal and perioperative intensive care management of patients with a single ventricle. BACKGROUND: Single-ventricle physiology is characterized by parallel pulmonary and systemic circulations, with effective blood flow to each determined by the relative resistances in the pulmonary and systemic vascular beds. Perinatal and perioperative management of these patients is largely based on empiric observations and differs considerably between institutions and is further complicated by the transitional physiology of the newborn. The lack of animal models of single-ventricle physiology has hindered the understanding of this problem. METHODS: A 10-mm, Damus-Kaye-Stansel-type aortopulmonary anastomosis was created in 10 fetal sheep at 140 +/- 1.2 days of gestation. The main pulmonary artery was ligated distally, and pulmonary blood flow (Qp) was provided through a 5-mm aortopulmonary shunt. Eight lambs were delivered at term and placed on cardiopulmonary bypass (30 min) 48 to 72 h after birth. Pharmacologic interventions (0.1 microgram/kg body weight per min of epinephrine, 2 mEq/kg of sodium bicarbonate and 10 mg/kg of calcium chloride) were performed before and after bypass, and hemodynamic responses were observed. The response to the epinephrine bolus was determined only in the postbypass study. RESULTS: Both before and after bypass, epinephrine infusion and calcium and bicarbonate administration increased Qp and systemic blood flow (Qs) (total cardiac output) but produced only small changes in the Qp/Qs ratio (-0.5% to -7.3% change). With the epinephrine bolus, Qp increased enormously, and the Qp/Qs ratio increased by 584% (p < 0.001). CONCLUSIONS: In neonatal lambs with single-ventricle physiology created in utero, epinephrine infusion and calcium and bicarbonate administration increased total cardiac output without significantly compromising the Qp/Qs ratio. However, epinephrine bolus seems to be hemodynamically detrimental in circumstances of single-ventricle physiology and should be used with caution and probably in relatively lower doses in the resuscitation of patients with single-ventricle physiology. Further investigation of the dose-dependent effects and the effects of prolonged administration of common pharmacologic agents will enable better management of patients with single-ventricle physiology.

Animals↗
PubMed1996

T cell development within the intestinal mucosa: clues to a novel immune-endocrine network?

Small intestine intraepithelial lymphocytes (IELs) comprise a heterogeneous and phenotypically complex population of T cells that are part of the gut-associated lymphoid tissues (GALTs). Recent studies from a number of laboratories indicate that murine IELs are greatly enriched for extrathymic T cells, although many aspects of the IEL extrathymic developmental pathway remain controversial, and there is currently no consensus of opinion as to which IELs are extrathymic and which are thymus-derived. Those differences reflect variations in the IEL repertoire in athymic animals depending upon the specific model used to study IELs, and they correlate with the age at which mice became or were rendered athymic, implying that the thymus participates either directly or indirectly in the local extrathymic IEL developmental process. In this article, the basic findings regarding intestinal T cell development are discussed, and a hypothesis is provided which links neuroendocrine interactions targeted to the intestine epithelium to the striking relationship between animal developmental age and the thymopoietic potential of the intestine.

Animals↗
PubMed1996

Hormone regulation of murine T cells: potent tissue-specific immunosuppressive effects of thyroxine targeted to gut T cells.

Recent studies in athymic mice indicate that the neuroendocrine hormones thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH) can significantly influence the development of lymphoid cells associated with intestinal intraepithelial lymphocytes (IEL). In the present study we have examined the effects of those hormones, as well as of thyroxine (T4), a thyroid-derived hormone regulated by TSH, on IEL development in euthymic mice. As reported here, whereas IEL in euthymic mice were unaffected by TRH and TSH treatment, T4 administered to adult euthymic mice at 3 or 6 weeks of age caused a dramatic reduction in the numbers of TCR alpha beta, CD8 alpha beta IEL, i.e. the same subsets previously shown to be up-regulated ty TRH and TSH in athymic mice. When given to euthymic mice >8 weeks of age, after TCR alpha beta and CD8 alpha beta subsets had reached normal levels, T4 had minimal effect on IEL, suggesting that the mode of action of T4 is directed to developing but not mature IEL. That possibility was confirmed in experiments in which T4 treatment of bone marrow radiation chimeras during an active phase of T cell regeneration temporarily halted all IEL development at a stage characteristic of immature IEL. Most interesting, the immunosuppressive effects of T4 were selectively targeted to the intestinal immune system since T4 had no effect on developing thymocytes or on mature peripheral T cells, in either normal euthymic mice or during hematopoietic reconstitution of radiation chimeras. These findings have implications for understanding intestinal immunity and disease, including chronic intestinal inflammation, in ways not previously appreciated.

Animals↗
PubMed1996

Fetal cardiac bypass using an in-line axial flow pump to minimize extracorporeal surface and avoid priming volume.

BACKGROUND: Progressive metabolic acidosis, hypoxia, and hypercarbia develop rapidly after fetal cardiac bypass mainly as a result of an increase in placental vascular resistance and a decrease in placental blood flow. A number of factors including fetal stress, priming substances, and extracorporeal surfaces have been identified as possible stimuli causing this placental dysfunction. The purpose of this study was to examine the effects of avoiding priming volume and minimizing extracorporeal surface area on placental hemodynamics and function. METHODS: Fetal sheep (n = 16) at 118 to 122 days of gestation were subjected to cardiac bypass for 30 minutes using either an in-line axial-flow pump (Hemopump group: n = 8, no prime) or a roller pump with a venous reservoir (control group: n = 8, priming volume = 150 mL). After bypass, the fetuses were observed for 90 minutes. Placental blood flow and combined ventricular output were continuously measured with ultrasonic flow probes, and fetal blood gases were measured at specific intervals. RESULTS: Three fetuses in the control group died during the study, whereas all 8 fetuses in the Hemopump group remained in stable condition throughout the study period. During and after bypass, placental blood flow was significantly higher (p < 0.0001) and placental vascular resistance was significantly lower (p < 0.0001) in the Hemopump group than in the control group. Arterial pH and partial pressure of arterial oxygen declined significantly less (p < 0.0001), and partial pressure of arterial carbon dioxide increased significantly less (p = 0.0002) in the Hemopump group than in the control group. CONCLUSIONS: Reducing the extracorporeal surface area and avoiding external priming substances preserves placental hemodynamics after fetal cardiac bypass. An in-line axial-flow pump is useful in miniaturizing the bypass circuits for potential use in fetal cardiac surgery.

Acidosis↗
PubMed1995

Cloning and characterization of brnQ, a gene encoding a low-affinity, branched-chain amino acid carrier in Lactobacillus delbrückii subsp. lactis DSM7290.

A gene (brnQ), encoding a carrier for branched-chain amino acids in Lactobacillus delbrückii subsp. lactis DSM7290 was cloned in the low-copy-number vector pLG339 by complementation of a transport-deficient Escherichia coli strain. The plasmid carrying the cloned gene restored growth of an E. coli strain mutated in 4 different branched-chain amino acid transport genes at low concentrations of isoleucine, and increased its sensitivity to valine. Transport assays showed that leucine, isoleucine and valine are transported by this carrier and that transport is driven by the proton motive force. Nucleotide sequence analysis revealed an open reading frame of 1338 bp encoding a hydrophobic protein of 446 amino acids with a calculated molecular mass of 47864 Daltons. The start site of brnQ transcription was determined by primer extension analysis using mRNA from Lactobacillus delbrückii subsp. lactis DSM7290. The hydropathy profile suggests the existence of at least 12 hydrophobic domains that probably form membrane-associated alpha-helices. Comparisons of the nucleotide sequence of brnQ from Lactobacillus delbrückii subsp. lactis DSM7290, the amino acid sequence of its product and the topology of the hydrophobic domains with those of the respective carrier genes and proteins of Salmonella typhimurium and Pseudomonas aeruginosa revealed extensive homology.

Amino Acid Sequence↗
PubMed1995

T cell precursors in the spleen give rise to complex T cell repertoires in the thymus and the intestine.

T cell precursors located in peripheral immune tissues have been studied according to the potential to repopulate the thymus and the gut of lethally irradiated mice. T cell repopulation could be achieved with spleen cells from athymic or euthymic mice thoroughly devoid of mature T cells. Repopulation did not occur with lymph node lymphocytes as determined from studies in congenic mice. The kinetics of T cell repopulation differed in the gut and thymus in that donor-derived T cells appeared in the gut by day 7 after cell transfer, and in the thymus by day 14 after cell transfer. The multipotent nature of splenic T cell precursors was evident from the finding that all major phenotypic subsets of T cells in the thymus and the gut developed after spleen cell transfer. T cell repopulation of the intraepithelial lymphocytes also occurred efficiently in athymic radiation chimeras injected with spleen cells from congenitally athymic nude mice, demonstrating that gut T cell repopulation by those cells does not require a functional thymus. PCR-spectratype analyses of twenty-four V beta TCR genes in thymocytes and intraepithelial lymphocytes revealed extensive TCR-beta repertoires in both tissues 1 to 2 wk after cell transfer, although T cells with rearranged TCR were undetectable in the donor spleen cell population. The minimal phenotype of the splenic T cell precursor was determined to be CD3-, CD4-, CD8-, HSA+.

Animals↗
PubMed1995

CD43 is a murine T cell costimulatory receptor that functions independently of CD28.

Costimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T cells, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28-independent fashion since the mAb costimulates T cells from the CD28-deficient mice as well as wild-type mice. Expression cloning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.

Animals↗
PubMed1995

Cloning and DNA sequence analysis of pepQ, a prolidase gene from Lactobacillus delbrueckii subsp. lactis DSM7290 and partial characterization of its product.

From a genomic library of Lactobacillus delbrueckii subsp. lactis (DSM7290) DNA, in the low-copy-number vector pLG339, a recombinant clone was selected, which complemented a mutation in the prolidase gene (pepQ) of Escherichia coli UK173. Nucleotide sequence analysis revealed an open reading frame of 1104 nucleotides corresponding to a protein of 368 amino acids with a calculated pI of 4.64 and a molecular mass of 41,087 Da. The start site of pepQ transcription was determined by primer extension analysis with mRNA prepared from L. delbrueckii. Based on homology of the gene product to various peptidases and on the substrate specificity determined, the peptidase was designated PepQ. The influence of various protease inhibitors and cations on peptidase activity indicated that PepQ is a metalloprotease. The absence of a membrane-spanning domain and a signal peptide sequence argues for a cytoplasmic localization of the enzyme.

Amino Acid Sequence↗
PubMed1995

Hormonal regulation of extrathymic gut T cell development: involvement of thyroid stimulating hormone.

We recently demonstrated that athymic mice which lack intraepithelial lymphocytes (IELs) considered to be thymus-dependent (TD) T cells develop those cells following exogenous treatment with the hypothalamic-derived neuropeptide, thyrotropin-releasing hormone (TRH). To explore the mechanism by which neuroendocrine (NE) hormones influence the development of IELs, we have studied the effects of thyroid-stimulating hormone (TSH), a pituitary hormone that is regulated by TRH, on IEL development in adult mice that were neonatally thymectomized (NTX). Adult NTX mice treated with exogenous TSH for 3 weeks developed normal proportions and numbers of TD (TCR alpha beta+, CD8 alpha beta+, CD5+, and Thy-1+) IELs in a manner similar to that of TRH-treated NTX mice. The effect of hormone therapy was long-lasting in that IEL subsets which developed during TSH treatment were present well after the known lifespan of TSH in vivo, and because the numbers of IEL remained higher than in untreated mice. These findings suggest that TD IELs develop extrathymically through a series of immunologic and hormonal interactions which are regulated by the thymus during some phase of embryonic life.

Animals↗
PubMed1995

Molecular cloning and DNA sequence analysis of pepL, a leucyl aminopeptidase gene from Lactobacillus delbrueckii subsp. lactis DSM7290.

A genomic library of Lactobacillus delbrueckii subsp. lactis DSM7290 DNA fragments from a Sau3A partial digestion in the low-copy-number vector pLG339, was used to screen Escherichia coli for the presence of peptidases. Using the chromogenic substrate leucine-beta-naphthylamide (Leu-NH-Nap) and E. coli strain CM89 lacking the corresponding enzyme activity in an enzymic plate assay, allowed the isolation of two peptidase genes; the newly described pepL and the recently cloned and sequenced pepN. Clones could be distinguished not only by the restriction pattern of isolated plasmids but also by the rate and intensity of their colour reaction with Leu-NH-Nap. Three out of five clones were identified to express the Lactobacillus pepN gene; the others were shown to express a second aminopeptidase gene, designated pepL. This gene, together with 200 bp upstream of the proposed AUG initiation codon, was further subcloned and sequenced. The corresponding open reading frame of 897 nucleotides is predicted to encode a protein of 299 amino acids (34,541 Da). Searching the EMBL database revealed similarity to the prolinase of Lactobacillus helveticus (45.8% identity), to the iminopeptidases of Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus (25.5%), and to the Bacillus coagulans prolinase (21.5%). Minor similarities were detected for hydrolytic enzymes with serine active sites. The product encoded by the pepL gene was functional but could not be visualized on Coomassie-blue-stained polyacrylamide gels. High level expression of peptidase L in E. coli was achieved by placing the gene under the control of the T7 promoter.

Amino Acid Sequence↗
PubMed1995

T-lymphopoietic capacity of the mouse intestinal epithelium.

Small intestine intraepithelial lymphocytes (i-IELs) comprise a heterogeneous population of T cells that are part of the gut-associated lymphoid tissues. Recent studies indicate that murine i-IELs are greatly enriched for extrathymic T cells. Yet there is disagreement as to which i-IELS are extrathymic and which are not, and many aspects of intestinal T-cell development remain controversial. In this article, the developmental lineages of murine i-IELs are examined, and the thymopoietic potential of the intestine epithelium is discussed. A mechanism is described whereby all intestinal T cells develop extrathymically, and a concept of intestinal T cells is presented which suggests that lineages of i-IELs need to be viewed according to developmental factors exclusive of thymus dependency.

Animals↗
PubMed1994

Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC.

A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-beta-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50,909 Da. The enzyme is functional and extremely overexpressed in E. coli.

Amino Acid Sequence↗
PubMed1994

Heat-stable antigen may be an early marker of extrathymic murine intestinal intraepithelial lymphocytes.

Using a system of bone marrow (BM) hematopoietic repopulation of irradiated euthymic and athymic mice, we have examined the early stages of extrathymic T-cell development within the murine small intestine epithelium. During a period of active extrathymic T-cell development, two distinct populations of intraepithelial lymphocytes (IEL) were present. One consisted of CD3+ lymphocytes with phenotypic properties of mature IEL. The other population consisted of a transient IEL subset that increased in abundance between days 5 and 14 post-BM transfer, and then declined. The majority of transient IEL in both types of mice expressed the heat-stable antigen, of which some cells coexpressed CD3 but were void of other markers common to mature T cells. Studies using freshly extracted IEL from normal nonirradiated mice found that approximately 3% to 5% of the IEL had phenotypic properties similar to the transient IEL observed during repopulation of radiation chimeras, indicating that such IEL are present within the gut epithelium of normal, nonirradiated mice. Identification of this IEL subset should greatly facilitate studies of extrathymic IEL development.

Animals↗
PubMed1994

Thymus-neuroendocrine interactions in extrathymic T cell development.

Studies of the development of murine intestinal intraepithelial lymphocytes (IELs) have yielded markedly different results depending on the experimental system used. In athymic radiation chimeras, IELs consist of all subsets found in euthymic mice; adult mice that were athymic at birth have only IELs that are positive for T cell receptor gamma delta and CD8 alpha alpha. These differences are resolved by the finding that administration of the neuropeptide thyrotropin-releasing hormone to adult mice thymectomized as neonates leads to the development of all IEL T cells. Thus, a neuroendocrine signal initiated by the thymus during fetal or neonatal life appears to be required for subsequent extrathymic maturation of gut alpha beta T cells.

Animals↗
PubMed1994

Determination and comparison of Lactobacillus delbrueckii ssp. lactis DSM7290 promoter sequences.

The transcriptional start points of ten Lactobacillus delbrückii ssp. lactis DSM7290 genes were determined by primer extension. The upstream located promoter regions, including potential -35 and -10 regions and the spacing between them were compared to the well-known Escherichia coli and Bacillus subtilis promoters. The Lb. delbrückii -35 consensus sequence (TTGACA) seems to be less conserved then the E. coli sequence. The nucleotides TGC were often found upstream of the -10 region (TATAAT). The most frequently observed spacing between the two core promoter regions was 17 nt and the main distance between the -10 region and the transcriptional start point was mostly determined to be 6 nt in contrast to 7 nt, as described for E. coli promoters. The preferred initiation nucleotides in Lb. delbrückii were shown to be definitely purines (A or G). The ribosome binding sites located downstream of the promoters revealed the consensus sequence 3'-UCCUCCU-5', being the predicted 3'-OH end of the Lactobacillus 16S rRNA with a high degree of homology to known 16S rRNAs.

Base Sequence↗