Immunohistochemical analysis of host reaction to heavyweight-, reduced-weight-, and expanded polytetrafluoroethylene (ePTFE)-based meshes after short- and long-term intraabdominal implantations.

BACKGROUND: Prosthetic meshes induce a variety of inflammatory changes in the host, which may lead to excessive scarring with detrimental clinical consequences, especially in the long term. This study aimed to characterize the degree of short- and long-term inflammatory changes induced by common prosthetic meshes. METHODS: Twenty 4 x 4-cm samples each of expanded polytetrafluoroethylene (ePTFE), heavyweight polypropylene (hPP), ePTFE/heavyweight polypropylene (ePTFE/hPP), and reduced-weight polypropylene/regenerated cellulose (rPP) were implanted intraperitoneally in 40 rabbits for 4 or 12 months. After explantation, samples of mesh/tissue complex were analyzed for the degrees of cellular apoptosis (enzyme-linked immunoassay [ELISA]) and cellular turnover (mouse monoclonal antibody). RESULTS: In the short term, the degree of apoptosis in the hPP mesh was significantly higher than in the ePTFE and rPP groups. Similarly, it was higher in the ePTFE/hPP group than in either the ePTFE or the rPP group. The amount of Ki-67-positive cells was significantly higher in the hPP group than in the ePTFE or rPP group. The cell turnover in the ePTFE/hPP group was similar to that in the hPP group, but significantly higher than in either the ePTFE or the rPP group. The rPP group, in turn, had a higher Ki-67 score than the ePTFE group. In the long term, both the degree of apoptosis and Ki-67 positivity were significantly lower in the rPP and ePTFE groups than in either the ePTFE/hPP or the hPP group. A significant decrease in Ki-67 scores between the short and long-term groups was found only in the rPP group. CONCLUSION: In the short term, heavyweight polypropylene-based meshes were associated with significantly higher cell proliferation and death. A significantly higher degree of apoptosis and cell turnover were associated with heavyweight polypropylene-based meshes even 1 year after implantation, indicating ongoing inflammation and scar remodeling. On the other hand, ePTFE and reduced-weight polypropylene meshes were associated with nearly physiologic levels of inflammatory markers. Overall, an exaggerated and persistent host foreign body response to heavyweight polypropylene-based meshes indicates poor biocompatibility, with potential detrimental clinical sequela.

In vitro infectability of prosthetic mesh by methicillin-resistant Staphylococcus aureus.

Although mesh use is important for effective herniorrhaphy in adults, prosthetic infections can cause serious morbidity. Bacterial adherence to the mesh is a known precursor to prosthetic infection. We compared the ability of common mesh prosthetics to resist bacterial adherence. The meshes studied included polypropylene (Marlex, expanded polytetrafluoroethylene (PTFE) with and without silver chlorhexidine coating (DualMesh Plus and Dualmesh) composite meshes (Composix E/X, Proceed, and Parietex Composite) and lightweight polypropylene meshes (TiMesh, Ultrapro, and Vypro). Fifteen samples of each mesh type were individually inoculated with a suspension of 10(8 )methicillin-resistant Staphylococcus aureus (MRSA) in tryptic soy broth. After incubation at 37 degrees C for 1 h, the mesh pieces were then removed and serially washed. The colony-forming units (CFU) of MRSA present in the initial inoculum, at the end of the 1-h warm-water bath (broth count), and the pooled washes (wash count), were determined using serial dilutions and spot plating. The bacteria not accounted for in the broth or wash counts were considered adhered to the mesh. Samples of each mesh type were also analyzed using scanning electron microscopy (SEM). Data are presented as the mean percentage adherence with ANOVA and Tukey's test used to determine significance (P<0.05). The DualMesh Plus mesh had no detectable MRSA in the broth or the pooled wash samples. Dualmesh had less adherence compared with Marlex, Proceed, and Vypro (P<0.05). Conversely, Vypro had a statistically higher adherence (96%, P<0.05) as compared to TiMesh, Ultrapro, Composix E/X, and Parietex Composite. SEM confirmed bacterial adherence to all the mesh types except DualMesh Plus. The ability of a biomaterial to resist infection has an important clinical significance. DualMesh Plus, due to its antimicrobial coating, is the only mesh type of the nine tested that demonstrated a bactericidal property. Standard PTFE (Dualmesh) also had less bacterial adherence. Vypro demonstrated an increase in bacterial adherence; this was possibly due to the multifilament polyglactin 910 weaved within the prolene component of the mesh.

Induction of globin synthesis in K562 cells is associated with differential expression of transcription factor genes.

Globin gene switching may be mediated by proteins expressed during different stages of development. Their identification may clarify the mechanisms of the conversion from fetal to adult globin production and lead to new approaches to reversing or retarding the gamma- to beta-globin gene switch. To explore this hypothesis, K562 erythroleukemia cells were induced to differentiate with 1.25, 2.5, and 5 mM sodium butyrate and gene expression was studied after 24, 48, and 72 h. Erythroid differentiation was verified by benzidine staining and by measuring the activity of a transduced A gamma-globin gene promoter linked to a luciferase reporter gene. Using differential display polymerase chain reaction (PCR), total mRNA extracted from induced cells at each time point of induction was reverse transcribed in the presence of A, G, and C anchored primers and 16 arbitrary primers, calculated to amplify approximately 50% of expressed genes. Amplified mRNAs from induced and uninduced cells were separated in polyacrylamide gels and compared. More than 110 cDNA fragments which appeared to represent either up- or downregulated mRNA species in induced K562 cells were identified. Sixty-four of these fragments had more than 95% homology to known GenBank sequences. Seventeen fragments with characteristics of transcription factors were cloned. These include differentiation-related gene-1 (drg-1), PAX 3/forkhead transcription factor, HZF2 which is a Kruppel-related zinc finger protein, three helix-loop-helix proteins (heir-1, Id3, and GOS8), alpha-NAC transcriptional coactivator, LIM domain protein, and trophoblast hypoxia regulating factor. Differential expression of all 17 fragments over 72 h was confirmed by reverse Northern dot blot analysis, semiquantitative PCR using nested primers, and Northern analysis. Erythroid maturation in induced K562 cells is associated with differential expression of numerous genes. Some encode transcription factors that could effect the initiation of HbF synthesis. Almost half of the differentially expressed clones contained cDNAs of unidentified open reading frames and these are the object of continued study.

Double heterozygosity for the codon beta 39 C-to-T nonsense mutation and a triplicate alpha-globin gene locus can cause "dominantly" inherited beta-thalassemia intermedia.

BACKGROUND: A beta-thalassemia intermedia phenotype can be caused by multiple genotypes. METHODS: We studied a family where the mother was hematologically normal and both father and daughter had beta-thalassemia intermedia. RESULTS: Both affected individuals were heterozygous for a codon 39 CAG-to-TAG mutation. They also were heterozygous for a triplicate alpha-globin gene locus (alphaalphaalpha(anti 3.7)). CONCLUSIONS: This compound heterozygous condition of a beta39 C-to-T mutation and triplicate alpha-globin gene increases alpha:beta-globin chain imbalance and accounts for the presence of beta-thalassemia intermedia. The proband received both an abnormal beta-globin gene and a triplicate alpha-globin locus from her father. Although the phenotype seems to be dominantly inherited, because of independent segregation of the alpha- and beta-globin genes, it is more accurately an example of polygenic inheritance.

Genetic studies suggest a multicentric origin for Hb G-Coushatta [beta22(B4)Glu-->Ala].

Hb G-Coushatta [beta22(B4)Glu-->Ala] is found in geographically separated ethnic groups. Commonest along the Silk Road region of China but also present in the North American Coushatta, we sought to determine whether this variant had a unicentric or multicentric origin. We examined the haplotype of the beta-globin gene cluster in two Chinese families and in five Louisiana Coushatta heterozygous for this mutation. Chinese and Louisiana Coushatta had different haplotypes associated with the identical Hb G mutation. These haplotypes were defined by the presence of a HindIII restriction site in the Agamma-globin gene and AvaII restriction site in the beta-globin gene in Chinese subjects and their absence in the Louisiana Coushatta. We found a CAC at codon beta2 (beta-globin gene framework 1 or 2) linked to the Hb G-Coushatta gene in Chinese, and a CAT (framework 3) in Louisiana Coushatta, indicating different beta-globin gene frameworks. Both the Hb G-Coushatta mutation (GAA-->GCA) and the codon 2 CAC-->CAT polymorphism are normal delta-globin gene sequences, suggesting the possibility of gene conversion. We conclude that Hb G-Coushatta had at least two independent origins. This could be due to separate mutations at codon beta22 in Chinese and Louisiana Coushatta, a mutation at this codon and a beta-->delta conversion, or two beta-->delta gene conversion events.

The validity of self-reported drug use data: the accuracy of responses on confidential self-administered answered sheets.

Official records offer a relatively inexpensive, nonintrusive strategy for checking on the accuracy of self-reported drug use. Responses of a small sample (N = 67) of former drug treatment clients interviewed using procedures exactly modeled on the National Household Survey on Drug Abuse were compared to their clinic records. The accuracy of reports compared to clinic records varied by drug, with the percentage of known users reporting their use highest for marijuana, followed by cocaine and hallucinogens, and lowest for heroin. Almost half of this sample of former treatment clients denied ever receiving drug treatment.

Beta-globin gene haplotype in Hb SC disease.

We asked the question, is the haplotype found with the sickle hemoglobin gene associated with different hematological characteristics in patients who were combined heterozygotes for sickle hemoglobin and hemoglobin C (Hb SC disease)? In 73 adults with Hb SC disease, a Benin haplotype chromosome was present in 56%, and Bantu (or Central African Republic; CAR), Senegal, and atypical haplotype chromosomes were found in 25%, 6%, and 12%, respectively. No significant difference were found in hematological characteristics or fetal hemoglobin levels of patients with Benin/C, CAR/C, Senegal/C, and atypical/C haplotypes. There were 71% C I, 18% C II, and 11% other beta(c) haplotypes. Fetal hemoglobin levels are lower in Hb SC disease than in sickle-cell anemia. Perhaps because haplotype has no discernible effect on fetal hemoglobin level in Hb SC disease, it does not modulate its hematological features.

Role of aldehyde oxidase in the in vitro conversion of famciclovir to penciclovir in human liver.

Famciclovir is the diacetyl 6-deoxy derivative of the active antiviral penciclovir that is for use in the treatment of infections caused by the herpes family of viruses. The major pathway of conversion is via di-deacetylation to BRL 42359, followed by oxidation to penciclovir. On oral dosing of famciclovir to humans, only penciclovir and BRL 42359 can be detected consistently in the plasma; thus, attention was focused on the oxidation reaction. This 6-oxidation occurred rapidly in human liver cytosol, had no requirement for cofactors, and followed simple Michaelis-Menten kinetics with a KM of 115 microM +/- 23 (N = 3). Using inhibitors of xanthine oxidase (allopurinol) and aldehyde oxidase (menadione and isovanillin), the relative roles of these enzymes in this process were determined. At a concentration of BRL 42359 that reflected plasma concentrations observed in humans (4 microM), both menadione (IC50 7 microM) and isovanillin (IC50 15 microM) caused extensive inhibition of the 6-oxidation reaction. In contrast, allopurinol caused no significant inhibition, confirming earlier in vivo work. At higher substrate concentrations (50 and 200 microM), the results with these inhibitors were broadly similar. These results provide strong evidence that aldehyde oxidase and not xanthine oxidase is responsible for the 6-oxidation of BRL 42359 to penciclovir in human liver cytosol, and this is likely to reflect the in vivo situation.

Two missense mutations in the beta-globin gene can cause severe beta thalassemia. Hemoglobin Medicine Lake (beta 32[B14]leucine-->glutamine; 98 [FG5] valine-->methionine).

We studied the molecular basis of transfusion-dependent hemolytic anemia in an infant who rapidly developed the phenotype of beta thalassemia major. DNA sequence of one beta-globin gene of the proband revealed two mutations, one for the moderately unstable hemoglobin (Hb) Köln and another for a novel codon 32 cytosine-thymidine-guanine-->cytosine-adenine-guanine transversion encoding a leucine-->glutamine mutation. A hydrophilic glutamine residue at beta 32 has an uncharged polar side chain that could potentially distort the B helix and provoke further molecular instability. This new hemoglobin was called Hb Medicine Lake. Biosynthesis studies showed a deficit of beta-globin synthesis with early loss of beta-globin chains. An abnormal unstable hemoglobin, globin chain, or tryptic globin peptide was not present, demonstrating the extreme lability of this novel globin. Hb Medicine Lake mRNA was present, but an aberrantly spliced message was not. Absence of an abnormal beta-globin gene in the mother makes it likely that a de novo mutation occurred in the proband. The molecular pathogenesis of Hb Medicine Lake illustrates a mechanism whereby the phenotype of a genetic disorder, like the mild hemolytic anemia associated with a hemoglobinopathy, can be modulated by a coincident mutation in the same gene.

Use of rat and human in vitro systems to assess the effectiveness and enzymology of deoxyguanine analogs as prodrugs of an antiviral agent.

BRL 55792, BRL 55791, and BRL 55039 are prodrugs of an active antiviral agent 9-(3-hydroxypropoxy) guanine (BRL 44385). The prodrugs were 6-deoxygenated analogs of BRL 44385, with ether groups substituted at the 9-position: BRL 55792 with an (isopropoxymethyloxy)propoxy group, BRL 55791 with a (methoxymethyloxy)propoxy group, and BRL 55039 with an ethoxypropoxy group. Conversion of the prodrugs to BRL 44385 had been demonstrated in vivo in the rat and involved 6-oxidation followed by dealkylation. Metabolism was studied in rat liver in vitro systems to find a model to evaluate BRL 44385 production. Rat hepatocytes performed both reaction steps and were used to assess which of the three prodrugs demonstrated greatest production of the active drug. BRL 55792 demonstrated greatest conversion in vitro, and this was in agreement with in vivo data. The production of BRL 44385 from BRL 55792 was also demonstrated in human hepatocyte incubations, providing evidence that these reactions can occur in humans, thereby increasing confidence that BRL 55792 would be suitable prodrug for human therapy. Further experiments were performed to investigate the enzymes involved in these conversions. The 6-oxidation step occurred in the cytosol. Use of allopurinol and menadione (xanthine and aldehyde oxidase inhibitors) indicated that these conversions were catalyzed exclusively by xanthine oxidase in the rat but mainly by aldehyde oxidase in humans. The dealkylation reaction was detected in hepatocytes but not in homogenates or subcellular fractions. Inhibition of this reaction by aminobenzotriazole and ketoconazole (P-450 inhibitors) indicated that it was mediated by cytochrome P-450.

Building a patient record system. New information technologies will support development of patient-focused care.

As part of its plan to build a regional integrated healthcare delivery network, Genesys Health System, Flint, MI, has been developing a computerized patient record (CPR). The CPR will give users throughout the system immediate access to diagnostic studies, treatment information, discharge summaries, operative reports, x-rays, and other patient information. Before considering technical aspects of the system, healthcare executives considering implementation of a CPR should examine business and clinical issues to determine what they want to accomplish with the CPR. The Genesys information system is being designed with the following goals in mind: Organizing based on patient needs; Allocating resources at the point of care; Working as a broadly skilled, empowered staff; Delegating authority and accountability; Using technology to enable patient-focused care within the context of the system vision. Genesys envisions a CPR system that brings together records from such sources as emergency rooms, outpatient clinics, community service organizations, physicians offices, care teams, managed care companies, and financial systems. Genesys's use of care plans for specified procedures and diagnoses will enable it to use exception-based documentation of care delivered, whereby only departures from the protocol or unexpected outcomes are recorded.

Use of rat and human in vitro systems to assess the effectiveness and enzymology of deoxy-guanine analogues as prodrugs of an antiviral agent.

BRL 55792, BRL 55791, and BRL 55039 are prodrugs of an active anti-viral agent 9-(3-hydroxypropoxy) guanine, (BRL 44385). The prodrugs were 6-deoxygenated analogues of BRL 44385 with ether groups substituted at the 9-position: BRL 55792 with an (isopropoxymethyloxy)propoxy group, BRL 55791 with a (methoxymethyloxy)propoxy group, and BRL 55039 with an ethoxypropoxy group. Conversion of the prodrugs to BRL 44385 had been demonstrated in vivo in rat and involved 6-oxidation followed by dealkylation. Metabolism was studied in rat liver in vitro systems to find a model to evaluate BRL 44385 production. Rat hepatocytes performed both reaction steps and were used to assess which of the three prodrugs demonstrated greatest production of the active drug. BRL 55792 demonstrated greatest conversion in vitro and this was in agreement with in vivo data. The production of BRL 44385 from BRL 55792 was also demonstrated in human hepatocyte incubations providing evidence that these reactions can occur in man thereby increasing confidence that BRL 55792 would be a suitable prodrug for human therapy. Further experiments were performed to investigate the enzymes involved in these conversions. The 6-oxidation step occurred in the cytosol. Use of allopurinol and menadione (xanthine and aldehyde oxidase inhibitors) indicated that these conversions were catalyzed exclusively by xanthine oxidase in the rat but mainly by aldehyde oxidase in man. The dealkylation reaction was detected in hepatocytes but not in homogenates or subcellular fractions. Inhibition of this reaction by aminobenzotriazole and ketoconazole (P-450 inhibitors) indicated that it was mediated by cytochrome P-450.

Beta-thalassemia in southwestern Iran.

Seventeen unrelated beta-thalassemia patients or carriers from Southwestern Iran were examined for the beta-globin gene mutations by polymerase chain reaction amplification of the beta-globin gene and direct genomic sequencing, or by the method of allele-specific oligonucleotide hybridization. Their clinical and hematological characteristics were also recorded. Of 26 potential thalassemia-causing chromosomes examined, 10 different mutations were found. The IVS-II-1 (G-->A) mutation was the most frequent (31%) followed by the IVS-I-6 (T-->C) mutation (15%). Eight mutations were initially described in Mediterranean populations and two were of Kurdish origin. Four of these mutations, both initially described in the Mediterranean region, are reported here for the first time in Iranians. The unexpectedly high number of different mutations that account for beta-thalassemia in this region of Iran suggest migration of chromosomes from distant places and genetic admixture.

Reporting of child maltreatment: a secondary analysis of the National Incidence Surveys.

Reports to CPS agencies of child maltreatment cases come from a variety of community sources. Which kinds of cases are underreported, overreported, or not reported at all, and why? We address these questions by examining the discrepancies between cases known to CPS agencies and those known to professionals who regularly come into contact with children: teachers, hospital personnel, law enforcement officers, court personnel, and social service workers. The analysis is based on the 1980 and 1986 National Study of the Incidence and Prevalence of Child Abuse and Neglect. Our research yielded three major findings. First, older victims were less likely than younger victims to be known to CPS agencies. Second, there is a hierarchy of type of abuse reported to CPS agencies, with sexual abuse being at the top of the list and educational neglect at the bottom of the list. Third, the victims' race, sex, and income did not play a role in whether or not a case was reported to CPS agencies.

G gamma A gamma (beta+) hereditary persistence of fetal hemoglobin: the G gamma -158 C-->T mutation in cis to the -175 T-->C mutation of the A gamma-globin gene results in increased G gamma-globin synthesis.

Hereditary persistence of fetal hemoglobin (HPFH) can be generally classified into deletional and nondeletional forms. The family described in the present study has characteristics of both types of HPFH. The proband is a healthy 30-year-old black woman. Analysis of her hemoglobin revealed 40.4% HbS, 40.9% HbF (G gamma/A gamma ratio 0.53), 16.8% HbA, and 1.9% HbA2. All of her hematologic indices were normal, and the distribution of HbF in her red cells was pancellular. Family studies demonstrated that the proband has one chromosome 11 bearing the beta s-globin gene and the other bearing a G gamma A gamma (beta+) HPFH determinant in cis to the beta A-globin gene. Gene mapping studies of the region between the G gamma- and beta-globin genes were normal. However, when the A gamma and G gamma promoters were amplified by polymerase chain reaction (PCR) and sequenced, the A gamma promoter was found to have the T-->C mutation at -175, and the G gamma promoter region was found to have the C-->T mutation at -158. The -158 C-->T mutation has been associated with elevated G gamma levels and high HbF in hemolysis, although its role in causing these effects is unclear. The present study suggests that this mutation can also enhance G gamma-globin expression in cis to the -175 T-->C mutation in the absence of hemolysis. We suggest that the alteration of the A gamma gene octamer binding site by the -175 mutation, as well as the loss of a putative G gamma "silencer" caused by the -158 mutation may account for this phenotype. We propose calling these linked mutations the G gamma A gamma(beta+) HPFH.

Evidence that famciclovir (BRL 42810) and its associated metabolites do not inhibit the 6 beta-hydroxylation of testosterone in human liver microsomes.

Famciclovir is the diacetyl 6-deoxy analog of the active antiviral compound penciclovir with potential use in the treatment of infections caused by the herpes family of viruses. The major pathway of metabolism of famciclovir is deacetylation to BRL 42359 followed by oxidation to penciclovir. It is possible that famciclovir may be coadministered with cyclosporin A to combat viral infections induced by immunosuppression in organ transplant and bone marrow transplant patients. As a result, information is required on possible interactions between the cytochrome P-450 3A substrate cyclosporin A and famciclovir and its metabolites in humans. In order to probe cytochrome P-450 3A activity, testosterone 6 beta-hydroxylation in two human liver microsomal preparations was measured. Nicardipine and ketoconazole, two drugs with known inhibitory interactions with cyclosporin A, were used as positive controls. Profiles of 6 beta-hydroxytestosterone production showed no inhibition effected by famciclovir, penciclovir, or BRL 42359 when marked inhibition was observed in incubations containing nicardipine, nifedipine, or ketoconazole. Further incubations of [14C]BRL 42359 with human liver cytosol and microsomes indicated that BRL 42359 is oxidized to penciclovir in cytosol but not in microsomes and that this reaction was not dependent on the presence of NADPH. Because P-450 resides mainly in the microsomal fraction and is dependent on the presence of cofactors for catalytic activity, it seems that this oxidation is not catalyzed by cytochrome P-450.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-thalassemia intermedia with exceptionally high hemoglobin A2: relationship to mutations in the beta-gene promoter.

Small deletions of the 5' portion of the beta-globin gene that remove the promoters but stop 3' to the delta-globin gene are recognized as the sole cause of beta-thalassemia with exceptionally high hemoglobin A2 (HbA2) levels. Two patients with beta-thalassemia intermedia and exceptionally high levels of HbA2 (10.4 and 12.0%) were examined. One patient was a combined heterozygote for the -88 C----T and a novel -87 C----A mutation, while the other was homozygous for the -29 A----G beta(+)-thalassemia mutation. The remainder of the beta genes were normal. There was no evidence for deletions involving the 5' portion of the beta gene or the region between the beta and delta genes. Gene mapping studies excluded the possibility of a beta delta-anti-Lepore hemoglobin gene with beta promoters and delta coding sequences. There were no mutations in the promoters of the G gamma or A gamma-globin genes that have been associated with the hereditary persistence of HbF phenotype. The delta-globin gene promoters were normal from codon 17 to position -145 relative to the mRNA capping site. There appears to be considerable heterogeneity of HbA2 and HbF levels in patients who are homozygous or mixed heterozygotes for mutations in the TATA box and other promoter elements of the beta-globin gene. The capacity for proteolysis within the erythrocyte may vary among individuals. The authors hypothesize that in the exceptionally high HbA2 beta-thalassemia intermedia phenotype, proteolysis of superfluous alpha-globin chains is less efficient than in patients with customary levels of HbA2.(ABSTRACT TRUNCATED AT 250 WORDS)