Sequestrated caudal catheter in a child: An anesthetic nightmare and surgical dilemma.

The usage of epidural infusion for intraoperative and postoperative pain relief is widely used in certain pediatric anesthetic practice because of the effectiveness and advantages. However, there is drawback for these techniques due to its potential complications such as inadvertent intrathecal placement, local anesthetic toxicity, catheter migration, infection, and breakage of epidural catheter. Though occur infrequently, epidural catheters have been known to snap during insertion or removal. The retained catheter tip may lead to multiple complications, including nerve injury, infection, and even catheter migration. Although there are literatures recommend options for management of removal of retained catheter, there are limited reports of these occurrences, especially among children. We report a case of sequestrated sheared epidural catheter segment in a child, aiming to share this experience for the future management of patients under similar condition.

Clinical Study to Evaluate the Association Between Sensorineural Hearing Loss and Diabetes Mellitus in Poorly Controlled Patients Whose HbA1c >8.

The relationship between sensorineural hearing loss (SNHL) and Diabetes mellitus has been known since more than 150 years. The pathophysiology of diabetes related hearing loss is speculative. Hearing loss is usually, bilateral, gradual onset, affecting higher frequencies. This study aims at knowing the prevalence of SNHL in DM and its relation to age, sex, duration of DM and control of DM. A total of 50 type 2 diabetics of age group 30-65 years were involved in the study. FBS, PPBS, HbA1c of all the subjects were done and later subjected to PTA. The type and severity of hearing loss was noted. Occurrence of SNHL was later compared with age, sex, duration, and control of DM. Sensorineural hearing loss was found in 66 % of type II diabetic patients and 34 % were found normal. Out of 50 diabetes mellitus patients, 33 patients had SNHL. All cases of SNHL detected were of gradual in onset and no one had hearing loss of sudden onset. Normal hearing was found in 34 % of patients, whereas 54 % of patients had mild hearing loss and 12 % of patients had moderate hearing loss. Association of hearing loss of DM patients with sex of the patient is insignificant. However there is significant association between older age group, longer duration and uncontrolled DM with that of SNHL. In subjects with HbA1c more than 8 and duration of diabetes mellitus more than 10 years prevalence of SNHL is more than 85 %, which is statistically significant. Sensorineural hearing loss in diabetes mellitus is gradually progressive involving high frequency thresholds. Hearing threshold increases with increasing age duration of diabetes and also high level of HbA1c greater than 8 %.

Transfusion transmitted infections in thalassaemics: need for reappraisal of blood screening strategy in India.

The aim of the study was to assess the blood safety in India through prevalence in thalassaemic population. Safety of the blood supply is a subject of great concern for all recipients. This review attempts to assess the relevance and format of tests for viruses in the context of transfusion transmitted infection (TTI) prevalence in India. Serological marker testing for human immunodeficiency virus-1/2 (HIV-1/2), hepatitis C virus (HCV) and hepatitis B virus (HBV) is mandatory in India. Numerous TTI incidents in the repeat recipients supported by results from nucleic acid technology (NAT) testing indicate the deficiencies in blood safety. The ß-thalassaemic population (3-17%) in India has been used to reflect on blood safety. The prevalence of HIV-1/2, HCV and HBV in the Indian donor population, the limitations in accessing safe donors, quality of serological tests and the impact on repeat recipients is evaluated. The reports point to prevalence of ˜2% of viral diseases in the blood donor population, and the insufficiency of serology testing resulting in up to 45% TTIs in thalassaemics. The revelation by individual donation (ID) NAT testing, of 1 per 310 units being serology negative-NAT reactive is alarming. Extrapolating the serology negative NAT reactive yields, for an annual blood supply of 7.9 million units, 23,700 units or nearly 100,000 blood components are likely to be infectious. Though the cost for ID-NAT testing is considered unaffordable for a medium development country such as India, the enormity of TTIs will place an unmanageable cost burden on the society.

Detection of West Nile virus RNA and antibody in frozen plasma components from a voluntary market withdrawal during the 2002 peak epidemic.

BACKGROUND: The US West Nile virus (WNV) epidemic in the summer and fall of 2002 included the first documented cases of transfusion-transmitted WNV infection. In December 2002, the FDA supported a voluntary market withdrawal by the blood banking community of frozen blood components collected in WNV high-activity areas. At the time, the prevalence of viremia and serologic markers for WNV in the blood supply was undefined. STUDY DESIGN AND METHODS: In collaboration with America's Blood Centers, 1468 frozen plasma components (of approx. 60,000 frozen units voluntarily withdrawn from the market) were selectively retrieved from the peak epidemic regions and season (June 23, 2002-September 28, 2002). These units were unlinked, subaliquoted, and tested by WNV enzyme immunoassays (EIAs; Focus Technologies and Abbott Laboratories) and nucleic acid amplification tests (NATs; Gen-Probe Inc. and Roche Molecular Systems). RESULTS: Of the 1468 EIA results from Abbott and Focus, 7 were anti-immunoglobulin M (IgM)- and anti-immunoglobulin G (IgG)-reactive by both assays, 8 and 1 were IgM-only-reactive, and 8 and 23 were IgG-only-reactive, respectively. NAT by Gen-Probe and Roche Molecular Systems yielded one RNA-positive, antibody-negative unit containing approximately 440 RNA copies per mL. An additional 10-fold replicate NAT testing by Gen-Probe on 14 of 15 IgM-reactive specimens yielded 2 additional IgM- and IgG-reactive units with low-level viremia (i.e., 7/10 and 2/10 replicates tested reactive). CONCLUSION: The prevalence of acute (RNA-positive) and recent (IgM-seroreactive) WNV infections indicates that transfusion risk in high-risk areas could have been considerable and that voluntary market withdrawal of frozen components likely averted some WNV transfusion transmissions. The existence of very-low-level viremic units raises concerns, because WNV minipool NAT screening will miss such units and individual NAT may not completely correct this situation.

Analytical and clinical sensitivity of West Nile virus RNA screening and supplemental assays available in 2003.

BACKGROUND: Transfusion-transmitted West Nile virus (WNV) infections were first reported in 2002, which led to rapid development of investigational nucleic acid amplification tests (NAT). A study was conducted to evaluate sensitivities of WNV screening and supplemental NAT assays first employed in 2003. STUDY DESIGN AND METHODS: Twenty-five member-coded panels were distributed to NAT assay manufacturers. Panels included five pedigreed WNV standards (1, 3, 10, 30, and 100 copies/mL), 15 or 16 donor units with very-low-level viremia identified through 2003 screening, and four or five negative control samples. Samples were tested neat in 10 replicates by all assays; for NAT screening assays, 10 replicates were also performed on dilutions consistent with minipool (MP)-NAT. The viral load distribution for 142 MP-NAT yield donations was characterized, relative to the analytical sensitivity of MP-NAT systems. RESULTS: Analytical sensitivities (50% limits of detection [LoD] based on Poisson model of detection of WNV standards) for screening NAT assays ranged from 3.4 to 29 copies per mL; when diluted consistent with MP pool sizes, the 50 percent LoD of screening NAT assays was reduced to 43 to 309 copies per mL. Analytical sensitivity of supplemental assays ranged from 1.5 to 7.7 copies per mL (50% LoD). Detection of RNA in donor units varied consistent with analytical LoD of assays. Detection of low-level viremia after MP dilutions was particularly compromised for seropositive units, probably reflecting lower viral loads in the postseroconversion phase. Based on the viral load distribution of MP-NAT yield donations (median, 3519 copies/mL; range, < 50-690,000), 13 to 24 percent of units had viral loads below the 50 percent LoD of screening NAT assays run in MP-NAT format. CONCLUSION: WNV screening and supplemental assays had generally excellent analytical sensitivity, comparable to human immunodeficiency virus-1 and hepatitis C virus NAT assays. The presence of low-level viremic units during epidemic periods and the impact of MP dilutions on sensitivity, however, suggest the need for further improvements in sensitivity as well as a role for targeted individual-donation NAT in epidemic regions.

Cloning of CCRL1, an orphan seven transmembrane receptor related to chemokine receptors, expressed abundantly in the heart.

In an attempt to identify novel chemokine receptor genes, cDNA expressed sequence tags (EST) were analyzed for a significant homology with mammalian chemokine receptors. The sequence from one of the selected EST clones was used to generate a full-length cDNA encoding a putative seven transmembrane receptor, CCRL1-CC chemokine receptor like 1. The full-length receptor encodes a polypeptide of 350 amino acids and has about 35% homology to the chemokine receptors CCR6 and CCR7. Northern blot analysis indicates predominant expression of about 5.0, 2.0 and 1.3kb mRNA forms in human heart tissue, while low-level expression of the 2.0 and 1.3kb forms was observed in lung, pancreas and spleen and in fetal tissues. In-situ hybridization confirmed the presence of CCRL1 mRNA in cardiac muscle cells. Similar to the chemokine receptor CCR6, CCRL1 maps to chromosome 6 and has one intron in the 5' untranslated region. Coupled transcription-translation of CCRL1 cDNA yielded a glycosylated polypeptide of about 45kDa.

Semirational design of a potent, artificial agonist of fibroblast growth factor receptors.

Fibroblast growth factors (FGFs) are being investigated in human clinical trials as treatments for angina, claudication, and stroke. We designed a molecule structurally unrelated to all FGFs, which potently mimicked basic FGF activity, by combining domains that (1) bind FGF receptors (2) bind heparin, and (3) mediate dimerization. A 26-residue peptide identified by phage display specifically bound FGF receptor (FGFR) 1c extracellular domain but had no homology with FGFs. When fused with the c-jun leucine zipper domain, which binds heparin and forms homodimers, the polypeptide specifically reproduced the mitogenic and morphogenic activities of basic FGF with similar potency (EC50 = 240 pM). The polypeptide required interaction with heparin for activity, demonstrating the importance of heparin for FGFR activation even with designed ligands structurally unrelated to FGF. Our results demonstrate the feasibility of engineering potent artificial agonists for the receptor tyrosine kinases, and have important implications for the design of nonpeptidic ligands for FGF receptors. Furthermore, artificial FGFR agonists may be useful alternatives to FGF in the treatment of ischemic vascular disease.

High-throughput screening for ligand-induced c-fos mRNA expression by branched DNA assay in Chinese hamster ovary cells.

We have developed a generally useful screening assay for receptor agonists and antagonists in Chinese Hamster Ovary (CHO) cells. Three key features of the assay make it applicable to a broad range of receptors: (1) the use of CHO cells as host cells to overexpress receptors, (2) measurement of endogenous c-fos mRNA, which responds to a wide spectrum of stimuli, and (3) the use of branched chain DNA assay which is highly sensitive, quantifiable, amenable to high-throughput analysis, and easy to execute. The combination of these features provides a powerful means to screen rapidly for peptide and small molecule ligands for a variety of receptors. CHO cells overexpressing insulin receptor were used as a test system to compare conventional signaling assays with the high-throughput c-fos branched DNA assay.

Interleukin-8 receptors R1 and R2 activate mitogen-activated protein kinases and induce c-fos, independent of Ras and Raf-1 in Chinese hamster ovary cells.

Many of the biological effects of interleukin-8 (IL-8) are realized by binding to the two seven-transmembrane receptors IL-8 R1 and IL-8 R2. IL-8 R1 is activated only by IL-8, while IL-8 R2 is activated by IL-8, GROalpha, and a few other alpha chemokines. In addition to the well-known chemoattractant function, IL-8 is also angiogenic and mitogenic. IL-8 R1 and R2 have been shown to interact with Galphai2 and Galpha16, resulting in the activation of several mitogen-activated protein kinases. We have investigated IL-8 R1 and IL-8 R2 regulated upstream mediators and downstream effects of extracellularly responsive kinase (ERK) signaling pathways by expressing the individual receptors in a heterologous system. Our results demonstrate the following in CHO cells stably expressing either IL-8 R1 or R2 receptors: (a) IL-8 activates ERK and ERK kinases (MEK) through R1. Both IL-8 and GROalpha activate ERK and MEK through R2, whereas MIP-1alpha, a beta chemokine, does not activate these kinases through either of these receptors. (b) ERK activation is inhibited by pertussis toxin and MEK1 inhibitor. (c) ERK activation is independent of the upstream mediators Ras and Raf-1. (d) The downstream effects of ERK activation result in an increase of c-fos mRNA through both R1 and R2 receptors.

Antibody testing and RT-PCR results in hepatitis C virus (HCV) infection: HCV-RNA detection in PBMC of plasma viremia-negative HCV-seropositive persons.

To evaluate the concordance between viremia and antibody testing in hepatitis C virus (HCV) diagnosis, 682 serum or plasma samples collected from patients with known or suspected HCV infection were tested. An overall concordance of 77% between serological and PCR results was found, 5% was RNA positive/antibody negative and 18% antibody positive/RNA negative. The relationship between HCV infection, risk group and clinical diagnosis was studied in 116 patients: the presence of anti-HCV antibody without viremia was shown in 72.7% of asymptomatic subjects and 17.6% of chronic hepatitis subjects without interferon treatment. However, the detection of HCV-RNA in peripheral blood mononuclear cells (PBMC) in four out of 38 plasma viremia-negative HCV-seropositive subjects (10.5%), showed that HCV-RNA could persist in PBMC and could begin the viral replication again at different times. The detection of HCV-RNA in PBMC in anti-HCV-positive subjects without viremia could reduce false-negative results of HCV-RNA testing by RT-PCR in serum or plasma.

Inhibition of adenylyl cyclase by alpha chemokines IL-8 and GRO-alpha in Chinese hamster ovary cells expressing R1 and R2 receptors.

Interleukin-8 (IL-8) and growth-related oncogene protein-alpha (GRO-alpha) belong to a family of alpha chemokines. The biologic effects of IL-8 are realized by binding to two seven-transmembrane receptors R1 and R2 and that of GRO-alpha by binding to receptor R2. Chinese hamster ovary (CHO) cells stably expressing R1 and R2 have been used to demonstrate that the ligand-dependent signaling by both receptors is via the inhibition of adenylyl cyclase. This inhibition is pertussis toxin sensitive and could be mediated by G(alpha)i2, which is present in CHO cells. GRO-alpha inhibits adenylyl cyclase exclusively in CHO-R2 cells, and IL-8 inhibits in both CHO-R1 and CHO-R2 cells. The cAMP status in cells is an easy, reliable, quantifiable signal that is amenable to high throughput screening for small molecule analogs.

Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by TRAF2.

Tumor necrosis factor alpha (TNF alpha) a pro-inflammatory cytokine is an endogenous mediator of septic shock, inflammation, anti-viral responses and apoptotic cell death. TNF alpha elicits its complex biological responses through the individual or cooperative action of two TNF receptors of mol. wt 55 kDa (TNF-RI) and mol. wt 75 kDa (TNF-RII). To determine signaling events specific for TNF-RII we fused the extracellular domain of the mouse CD4 antigen to the intracellular domain of TNF-RII. Crosslinking of the chimeric receptor using anti-CD4 antibodies initiates exclusively TNF-RII-mediated signals. Our findings show that: (i) TNF-RII is able to activate two members of the MAP kinase family: extracellular regulated kinase (ERK) and c-jun N-terminal kinase (JNK); (ii) TRAF2, a molecule that binds TNF-RII and associates indirectly with TNF-RI, is sufficient to activate JNK upon overexpression; (iii) dominant-negative TRAF2 blocks TNF alpha-mediated JNK activation and (iv) TRAF2 signals the activation of JNK and NF-kappaB through different pathways. Our findings suggest that TNF alpha-mediated JNK activation in fibroblasts is independent of the cell death pathway and that TRAF2 occupies a key role in TNF receptor signaling to JNK.

Receptor recognition and specificity of interleukin-8 is determined by residues that cluster near a surface-accessible hydrophobic pocket.

To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO gamma, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity. Receptor binding activity was tested by competition of 125I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines. Substitution into IL-8 of the GRO gamma sequences corresponding to either the amino-terminal loop (amino acids 1-18) or the first beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2. The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2. Exchanges of the second beta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (amino acids 53-72) had no significant effect. When IL-8 sequences were substituted into GRO gamma, a single domain containing the second beta-sheet of IL-8 (amino acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2. The amino-terminal loop (amino acids 1-18) and the third beta-sheet (amino acids 46-53) of IL-8 had little effect when substituted individually but showed increased binding to both receptors when substituted in combination. Individual amino acid substitutions were made at positions where IL-8 and GRO gamma sequences differ within the regions of residues 11-21 and 46-53. IL-8 mutations L49A or L49F selectively inhibited binding to IL8R1. Mutations Y13L and F21N enhanced binding to IL8R1 with little effect on IL8R2. A combined mutation Y13L/S14Q selectively decreased binding to IL8R2. Residues Tyr13, Ser14, Phe21, and Lys49 are clustered in and around a surface-accessible hydrophobic pocket on IL-8 that is physically distant from the previously identified ELR binding sequence. A homology model of GRO gamma, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GRO gamma. These studies suggest that the surface hydrophobic pocket and/or adjacent residues participate in IL-8 receptor recognition for both IL8R1 and IL8R2 and that the hydrophobic pocket itself may be essential for IL8R1 binding. Thus this region contains a second site for IL-8 receptor recognition that, in combination with the Glu4-Leu5-Arg6 region, can modulate receptor binding affinity and IL8R1 specificity.

Natural interferon-alpha versus its combination with 6-methyl-prednisolone in the therapy of type II mixed cryoglobulinemia: a long-term, randomized, controlled study.

Type II mixed cryoglobulinemia (MC) is an often progressive vasculitis characterized by circulating cold-precipitable proteins that usually consists of polyclonal IgG and monoclonal IgM kappa with rheumatoid factor (RF) activity. Its etiology is unknown, although recent evidence strongly suggests that hepatitis C virus (HCV) plays a major role. Plasmapheresis, corticosteroids, and cytotoxic drugs have been used in the therapy of MC patients. Recently, favorable results with recombinant interferon-alpha (rIFN alpha) have been reported. To further assess its effectiveness, we studied the effects of natural human interferon-alpha (nIFN alpha), alone and in combination with 6-methyl-prednisolone (PDN), in a prospective, randomized, controlled trial in patients with symptomatic MC. Sixty-five patients were enrolled onto the trial, 52 (80%) of whom presented serum anti-HCV antibodies and specific genomic RNA sequences. Fifteen patients received nIFN alpha (3 MU) intramuscularly (IM) three times weekly, whereas 17 patients also received 16 mg/d of PDN orally on non-IFN days. Moreover, 18 patients received 16 mg/d of PDN only, and 15 were untreated. Treatment was discontinued after 1 year and patients were monitored for 8 to 17 months (mean, 13). A complete response was achieved in eight of 15 patients (53.3%) treated with nIFN alpha and nine of 17 (52.9%) treated with nIFN alpha plus PDN, as compared with three of 18 patients (16.7%) who received PDN only (P < .05) and one of 15 (6.7%) untreated controls (P < .01). Partial response occurred in two of 15 (13.3%) patients treated with nIFN alpha, three of 17 (17.6%) who received nIFN alpha plus PDN, one of 18 (5.5%) who received PDN only, and one of 15 (6.7%) controls. A complete response in six patients (66.7%) was achieved within 3 months in the group that received nIFN alpha plus PDN, as compared with two patients (25%) of those who received nIFN alpha alone (P < .02). In anti-HCV-positive patients, the clinical response occurred in step with reduced or undetectable levels of HCV RNA and transaminase normalization. Quantification of circulating HCV RNA represented a good predictive response marker. The probability of relapse within 3 months after treatment was 100% (three of three patients) and 75% (six of eight patients), respectively, in patients who received PDN alone or nIFN alpha alone as compared with none of those who received nIFN alpha plus PDN (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Two distinct human endothelin B receptors generated by alternative splicing from a single gene.

A novel variant of endothelin B receptor (ETB) has been found in human brain, placenta, lung, and heart by reverse transcriptase polymerase chain reaction. This variant ETB1 has an additional 30 nucleotide sequence with splice sites at both ends. This results in a 10 amino acid increase in the length of the second cytoplasmic domain of ETB. Polymerase chain reaction on genomic DNA indicates that this sequence is part of the 134 bp intron which separates the second and third exons and is contiguous with the third exon of the ETB gene. Southern blot analysis of chromosomal DNA and genomic PCR results indicate that ETB1 arises by alternative RNA splicing of the single copy ETB gene. The insert sequence in ETB1 gene is absent in bovine, rat, and porcine DNA, and is unique to human DNA. Both ETB and ETB1 have been expressed in heterologous systems to examine their ligand binding and functional properties. Reverse transcriptase polymerase chain reaction of RNA from ETB1 expressing cells indicates that the additional sequence is stably expressed.

Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) and Genome Walking.

The polymerase chain reaction (PCR) is used for selective amplification of DNA fragments from both prokaryotes and eukaryotes (1-3). The only requirement for amplification is that the sequence of the extremities of the DNA fragment to be amplified be known (4). This places a limitation on the use of PCR in the amplification of adjacent unknown regions. We have developed a method that allows the amplification of double-stranded DNA even when the sequence information is available at one end only (5). This method, the single specific primer-PCR (SSP-PCR), permits amplification of genes for which only a partial sequence information is available, and allows unidirectional genome walking from known into unknown regions of the chromosome.

Antimalarial activity of optical isomers of quinacrine dihydrochloride against chloroquine-sensitive and -resistant Plasmodium falciparum in vitro.

Both enantiomers of quinacrine and the racemic form of the drug showed equal activity in vitro against chloroquine-sensitive and -resistant strains of Plasmodium falciparum, without detectable stereoselectivity. This contrasts with observations on chloroquine, where a similar lack of stereoselectivity in vitro is accompanied by a 10-fold loss of activity against the resistant strain. The observed in vivo differences reported for the enantiomers of chloroquine and the observations on the optically active metabolites of chloroquine and quinacrine may therefore be ascribed to a difference in the pharmacokinetics of their enantiomers.