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Biomedical subjects

M B Klein

Publications and source records attributed to M B Klein.

At least 19 recordsLinked to original sources
PubMed2005

Validation of a simple model for predicting liver fibrosis in HIV/hepatitis C virus-coinfected patients.

OBJECTIVES: Recently, several models incorporating laboratory measurements have been validated for use as surrogate markers for liver fibrosis in hepatitis C virus (HCV) mono-infection, the simplest of these being the aspartate aminotransferase (AST) to platelet ratio index (APRI). We evaluated how well the APRI predicts significant hepatic fibrosis in patients with HIV/HCV coinfection. METHODS: Forty-six HIV/HCV-coinfected patients who underwent liver biopsy and had concomitant laboratory measurements (+/-3 months) were included in the study. Significant fibrosis was defined as F2-F4 using Batt and Ludwig scoring (=3 Ishak). APRI=[(AST/upper limit of normal)/platelet count (10(9)/L)] x 100. We used sas proc logistic (SAS Institute, Cary, NC) to calculate the area under the receiver operating curve (ROC) (AUC). Sensitivities, specificities, positive predictive value (PPV) and negative predictive value (NPV) were compared using cut-offs previously identified in the literature. RESULTS: Thirty-three of 46 patients (72%) had significant fibrosis on biopsy. For significant fibrosis, the area under the ROC for the APRI was 0.847+/-0.057. APRI scores >1.5 (the higher cut-off) were 100% specific and 52% sensitive; PPV was 100% and NPV 45%. Scores <0.5 (the lower cut-off) were 82% sensitive and 46% specific in ruling out significant fibrosis (PPV 79%; NPV 50%). CONCLUSIONS: A simple model incorporating readily available laboratory data is highly predictive of significant fibrosis in HIV/HCV coinfection and could serve as a biopsy-sparing measure, thus making treatment more accessible for this population.

Adult↗
PubMed2004

CT angiography in complex upper extremity reconstruction.

Computed tomography angiography is a new technique that provides high-resolution, three-dimensional vascular imaging as well as excellent bone and soft tissue spatial relationships. The purpose of this study was to examine the use of computed tomography angiography in planning upper extremity reconstruction. Seventeen computed tomography angiograms were obtained in 14 patients over a 20-month period. All studies were obtained on an outpatient basis with contrast administered through a peripheral vein. All the studies demonstrated the pertinent anatomy and the intraoperative findings were as demonstrated in all cases. Information from two studies significantly altered pre-operative planning. The average charge for computed tomography angiography was 1,140 dollars, compared to 3,900 dollars for traditional angiography.

Adolescent↗
PubMed2001

Flexor tendon wound healing in vitro: the effect of lactate on tendon cell proliferation and collagen production.

Flexor tendon repair in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. Lactate is an early mediator of wound healing known to play an important role in stimulation of collagen production after cellular injury. Little attention has been paid to the role of lactate in flexor tendon wound healing. In this study tendon and tendon sheath were excised from rabbit forepaws. We examined proliferation of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes; collagen production by each of these 3 cell types; and effects of lactate on cell proliferation and collagen production. Three cell lines, tendon sheath, epitenon, and endotenon, were isolated and cultured. Tendon sheath fibroblasts showed the greatest proliferation. All 3 cell lines produced collagen I, II, and III. Lactate significantly increased collagen production by all 3 cell lines. We show that cells of the tendon sheath, epitenon, and endotenon produce collagen in vitro. Modulation of lactate levels may provide a means to modulate collagen production.

Animals↗
PubMed2000

The agent of human granulocytic ehrlichiosis induces the production of myelosuppressing chemokines without induction of proinflammatory cytokines.

Infection by human granulocytic ehrlichiosis (HGE) is characterized clinically by cytopenias out of proportion to the number of cells seen to be infected directly. To study the pathogenic role of inflammatory mediators in HGE infection, cytokine production by untreated and dimethyl sulfoxide-treated HL-60 cells, which demonstrate enhanced infection because of granulocytic differentiation, and by normal bone marrow cells was measured using modified sandwich ELISA assays on samples obtained sequentially after inoculation with the HGE agent. All infected cells produced physiological concentrations of CC (monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha and -beta, and RANTES) and CXC (interleukin [IL]-8) chemokines in amounts significantly greater than those produced by uninfected controls. In contrast, infected cells did not secrete the classic proinflammatory cytokines IL-1, IL-6, or tumor necrosis factor-alpha. The striking production of chemokines, powerful leukocyte chemoattractants capable of suppressing hematopoiesis, by susceptible target cells, is likely to be of pathogenic importance both in the observed cytopenias and in mediation of inflammation and host defenses during infection.

Bone Marrow Cells↗
PubMed2000

Management of hand and upper-extremity infections in heart transplant recipients.

Hand and upper-extremity infections are routinely managed by antibiotics, immobilization, and limited incision and drainage. However, in immunocompromised patients, these infections may be more aggressive and they may require more emergent treatment. The authors performed a retrospective review of the Stanford University Medical Center experience with hand and upper-extremity infections in 911 cardiac transplant recipients over the past 30 years. Thirteen heart transplant recipients were treated for infections of the hand and upper extremity on an inpatient basis. Ten patients (77 percent) required operative debridement, and three (23 percent) required more than one operative procedure. Nine patients (69 percent) had bacterial infections, six (46 percent) had fungal infections [four of these patients (31 percent) had both bacterial and fungal infections], one (7.7 percent) had a mycobacterial infection, and one (7.7 percent) was not cultured. Hand and upper-extremity infections in transplant recipients frequently resulted in deep-space infections, tenosynovitis, and osteomyelitis. The authors believe such infections represent a surgical emergency, requiring immediate evaluation by hand surgeons and early, aggressive treatment.

Adult↗
PubMed2000

Induction of tissue factor procoagulant activity in myelomonocytic cells inoculated by the agent of human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is a recently recognized rickettsial tick-borne febrile illness that may occasionally be complicated by coagulopathy. The agent of HGE (aHGE) is an obligate intracellular pathogen, which replicates in endosomes within neutrophils and their precursors. We hypothesized that aHGE might cause DIC via induction of monocyte tissue factor procoagulant activity (TF PCA). Peripheral blood mononuclear cells (PBMNC) and HL-60 cells were used to model the effect of aHGE infection on monocytes/macrophages. Mononuclear cells inoculated with aHGE in vitro demonstrated approximately a 12-15-fold increase in TF PCA, with peak activity occurring at 8-12 h. HL-60 cells inoculated with aHGE also manifested a 4-6 fold induction of TF PCA, with maximal activity occurring at about 8 h. By comparison, E. Coli lipopolysaccharide (LPS) also induced an increase in TF PCA of an equivalent magnitude, and with a similar time course. Induction of TF did not require inoculation of HL-60 cells with live organism, since heat-inactivated aHGE still stimulated TF PCA expression in the target cells. Furthermore, filtered supernatants from heat-inactivated organisms induced TF PCA suggesting that the effect is due to a soluble mediator produced by the organism. Although aHGE is a gram negative organism, the soluble mediator did not appear to be classic endotoxin in that the supernatants tested negative for endotoxin by the Limulus Amoebocyte assay, and polymixin had no inhibitory effect on aHGE supernatants. We conclude that aHGE induces cells of the myelo-monocytic lineage to synthesize TF, which may contribute to the clinical coagulopathy that can be observed in this condition. An atypical soluble mediator or cellular component of the organism appears to be critically important in TF induction by aHGE.

Blood Coagulation↗
PubMed1999

A cloned CD15s-negative variant of HL60 cells is deficient in expression of FUT7 and does not adhere to cytokine-stimulated endothelial cells.

The initial steps of leukocyte adhesion depend on selectin/ligand interactions. Surface ligands on leukocytes are often modified by addition of the sialyl Lewis x (CD15s) determinant. Biosynthesis of CD15s is dependent upon alpha(2,3)sialyltransferases and alpha(1,3)fucosyltransferases. We report the isolation of an HL60 cell line variant, HL60A2, that no longer expresses CD15s. HL60A2 cells do not adhere to cytokine-stimulated endothelial cells. Enzymatic assays reveal that this cell line has normal alpha(2,3)sialyltransferase activity but is deficient in the alpha(1,3)fucosyltransferase responsible for biosynthesis of CD15s (FUT7). The fucosyltransferase that constructs the non-sialylated antigen, Lewis x (CD15), is expressed at high levels (FUT4). Transcript analyses show that FUT7 and FUT4 are inversely expressed in HL60 and variant cell lines. HL60A2 cells provide a tool to study the regulation of selectin ligands and corresponding human fucosyltransferase genes.

Animals↗
PubMed1999

Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand.

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne illness caused by an intracellular bacterium that infects neutrophils. Cells susceptible to HGE express sialylated Lewis x (CD15s), a ligand for cell selectins. We demonstrate that adhesion of HGE to both HL60 cells and normal bone marrow cells directly correlates with their CD15s expression. HGE infection of HL60 cells, bone marrow progenitors, granulocytes, and monocytes was blocked by monoclonal antibodies against CD15s. However, these antibodies did not inhibit HGE binding, and anti-CD15s was capable of inhibiting the growth of HGE after its entry into the target cell. In contrast, neuraminidase treatment of HL60 cells prevented both HGE binding and infection. A cloned cell line (HL60-A2), derived from HL60 cells and resistant to HGE, was deficient in the expression of alpha-(1, 3)fucosyltransferase (Fuc-TVII), an enzyme known to be required for CD15s biosynthesis. Less than 1% of HL60-A2 cells expressed CD15s, and only these rare CD15s-expressing cells bound HGE and became infected. After transfection with Fuc-TVII, cells regained CD15s expression, as well as their ability to bind HGE and become infected. Thus, CD15s expression is highly correlated with susceptibility to HGE, and it, and/or a closely related sialylated and alpha-(1,3) fucosylated molecule, plays a key role in HGE infection, an observation that may help explain the organism's tropism for leukocytes.

Ehrlichia chaffeensis↗
PubMed1998

Critical role of phosphagens in the energy cascade of cutaneous ischemia and protective action of phosphocreatine analogues in skin flap survival.

A general understanding of the pivotal role of phosphocreatine (PCr) as the principal determinant of skin flap survival is now emerging. Definitive metabolic investigations using phosphorus (31P) and proton (1H) magnetic resonance spectroscopy (MRS) have established that the inability to replenish metabolically exhausted PCr reserves predictably correlates with skin flap necrosis. Furthermore, postoperative parenteral administration of PCr has been shown to augment effectively skin flap survival. We hypothesized that creatine kinase, the enzyme controlling the utilization of the high-energy phosphate component of PCr, is a critical determinant of the tolerance of a skin flap to ischemic insult. In other words, if the rate of utilization of PCr is too rapid, PCr stores will rapidly deplete, and the flap will not be able to withstand a period of ischemia. Alternatively, if the rate of dephosphorylation of PCr is reduced, survival of skin flaps during periods of ischemia could be extended. To test this hypothesis, we investigated the metabolic distribution and fate of cyclocreatine (cCr), a competent creatine analogue with a lower affinity for the creatine kinase enzyme. When administered as 1.5 percent (w/w) of the normal diet of laboratory rats, cCr accumulates in skin as the competent phosphagen, phosphocyclocreatine (PcCr). Cutaneous flaps elevated in these animals, and studied by 31P and 1H MRS, demonstrate that once depletion of PCr has occurred, PcCr continues to sustain ATP levels. This results in significant enhancement of skin flap survival (p < 0.005). These observations confirm the importance of the creatine kinase enzyme in cutaneous flap ischemia and suggest new approaches to augment skin flap survival.

Adenosine Triphosphate↗
PubMed1998

Monocytic differentiation inhibits infection and granulocytic differentiation potentiates infection by the agent of human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne infection with a specific tropism for granulocytes. We previously isolated and cultivated the HGE agent in the promyelocytic leukemia cell line HL-60 and have also demonstrated the susceptibility of both granulocytic and monocytic human marrow progenitors. Circulating monocytes have not been observed to be infected, suggesting that cell susceptibility may be differentiation specific. To evaluate this hypothesis, HL-60 cells were differentiated towards granulocytes (with dimethyl sulfoxide or all-trans retinoic acid) or toward monocytes-macrophages (with 12-O-tetradecanoylphorbol-13-acetate [TPA], gamma interferon, or 1, 25-dihydroxyvitamin D3) and then challenged with HGE. HGE binding, internalization, and proliferation were compared in differentiated and untreated control HL-60 cells by immunofluorescence, electron microscopy, and Giemsa staining. Granulocytic differentiation resulted in a doubling of HGE binding and enhanced infection consistent with the agent's clinical tropism for neutrophils. Granulocytic cells were unable to kill internalized ehrlichiae even after activation induced by N-formyl-Met-Leu-Phe alone or together with tumor necrosis factor alpha. In contrast, monocyte-macrophage differentiation with TPA resulted in complete resistance to infection through at least two distinct mechanisms: (i) reduction in binding and uptake and (ii) killing of any internalized organisms. Diminished binding in TPA-treated cells correlated with their reduced expression of sialyl Lewis x (CD15s), a putative cellular receptor component for HGE. The degree of monocytic differentiation and activation induced (i.e., TPA > gamma interferon > vitamin D3) correlated with resistance to HGE. Thus, HL-60 cells exhibit a striking differentiation-specific susceptibility to HGE. Differentiation-induced changes in bacterial adhesion and killing capacity underlie the tropism of HGE for granulocytic HL-60 cells and, conversely, the resistance of activated macrophages to infection.

Cell Differentiation↗
PubMed1997

Primary bone marrow progenitors of both granulocytic and monocytic lineages are susceptible to infection with the agent of human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne infection resulting in an acute febrile illness associated with cytopenias and characteristic intracellular organisms within peripheral blood granulocytes. The etiologic agent of HGE has recently been isolated and cultivated in the HL-60 promyelocytic leukemia cell line, but the spectrum of host cells that it naturally infects remains unknown. To determine if normal hematopoietic progenitors could be targets of infection, CD34+ primary human bone marrow cells, stimulated to differentiate along myelomonocytic lineages, were incubated with the HGE agent. Immature marrow progenitors and, remarkably, not only granulocytic but also CD14+ monocytic cells from these cultures supported replication of the HGE agent, suggesting that all are potential targets of infection in vivo. Infection of bone marrow progenitors may contribute to the hematologic manifestations of HGE. Furthermore, the ability of the agent to interact with monocytes has significant implications regarding disease pathogenesis and host response.

Cells, Cultured↗
PubMed1997

Antibiotic susceptibility of the newly cultivated agent of human granulocytic ehrlichiosis: promising activity of quinolones and rifamycins.

Human granulocytic ehrlichiosis (HGE) is a rapidly emerging tick-borne infection which presents as an acute febrile illness and is associated with hematologic abnormalities, elevated hepatic transaminase levels, and characteristic intracellular organisms in peripheral blood granulocytes. Although HGE has been successfully treated with tetracyclines, its susceptibility to other antibiotics remains unknown. No clear treatment alternative exist for young children, pregnant women, or allergic individuals, in whom tetracyclines are contra-indicated. We performed in vitro antibiotic susceptibility tests with this recently isolated agent grown in the human promyelocytic leukemia cell line HL-60. Doxycycline (MIC, 0.25 micrograms/ml), rifampin (MIC, 0.5 micrograms/ml), rifabutin (MIC, < or = 0.125 micrograms/ml), ciprofloxacin and ofloxacin (both with MICs of 2 micrograms/ml), and trovafloxacin (MIC, < or = 0.125 micrograms/ml) ciprofloxacin and ofloxacin (both with MICs of 2 micrograms/ml), and trovafloxacin (MIC, < or = 0.125 micrograms/ml) demonstrated significant activity against the HGE agent. These agents were also bactericidal. The HGE agent was resistant to clindamycin, trimethoprim-sulfamethoxazole, and imipenem-cilastatin, as well as to ampicillin, ceftriaxone, erythromycin, and azithromycin, antibiotics commonly used to treat Lyme disease. Both chloramphenicol and gentamicin had weak inhibitory activities but were not bactericidal. Our findings confirm the observed clinical efficacy of doxycycline and further suggest that the rifamycins and quinolones, particularly trovafloxacin, hold promise as alternative agents for treating this new infection.

4-Quinolones↗
PubMed1992

Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast.

The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.

Alleles↗
PubMed1989

Molecular cloning and primary structure of the uracil-DNA-glycosylase gene from Saccharomyces cerevisiae.

The structural gene for the Saccharomyces cerevisiae repair enzyme uracil-DNA-glycosylase (UNG1) was selected from a yeast genomic library in the multicopy vector YEp24 by complementation of the ung1-1 mutant in in vitro enzyme assays. The sequenced gene has an open reading frame which codes for a protein with molecular weight of 40,471. The measured size of the mRNA of 1.25 kb is in agreement with the predicted molecular weight of the protein. The gene product was overproduced about 100-fold in strains carrying an UNG1 gene containing plasmid at 100-200 copies/cell. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cleared lysates from such an overproducing strain, followed by renaturation of enzyme activity from individual gel slices showed the presence of two enzymatic activities in comparable quantities with Mr values of 39,500 and 33,000, indicating that the full size protein is either readily degraded in vivo or is very sensitive to proteolytic digestion in vitro. The carboxyl-terminal two-thirds of the yeast uracil-DNA-glycosylase is highly homologous to the entire Escherichia coli enzyme (50% amino acid identity). Genetic mapping experiments have localized the UNG1 gene on the left arm of chromosome XIII at 17 cM from the GAL80 locus proximal to the centromer. Deletions of the UNG1 gene are viable.

Amino Acid Sequence↗
PubMed1986

Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase.

A coliphage M13 chimer containing the Saccharomyces cerevisiae TRP1 gene and ARS1 replication origin (mPY2) was grown on an ung- dut- strain of Escherichia coli. The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil. This DNA failed to transform a delta trp1 yeast strain to prototrophy. However, when a mutagenized yeast stock was transformed with uracil-containing single-stranded mPY2 DNA, unstable transformants were obtained. After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA. In vitro, these mutants were defective for uracil-DNA-glycosylase activity. They were designated ung1. Strains containing the ung1 mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool. They have a moderate mutator phenotype for nuclear but not for mitochondrial genes. A low mitochondrial uracil-DNA-glycosylase activity was demonstrated in the mutant strains.

Cell Nucleus↗