Routine blood monitoring in maintenance immunoglobulin treatment of inflammatory neuropathy: Is it clinically relevant?

BACKGROUND: Pre-treatment screening for IgA deficiency and close monitoring of full blood count(FBC) and renal function is recommended with intravenous immunoglobulin(IVIg) therapy in neurological diseases. AIMS: To examine the frequency of biochemically defined and clinically significant episodes of treatment associated haemolysis, neutropenia, thrombocytopenia and acute kidney injury(AKI) in a cohort of patients on maintenance Immunoglobulin(Ig) therapy for inflammatory neuropathy. METHODS: A retrospective review of routine blood monitoring in patients from two UK specialist peripheral nerve centres. Accepted definitions for clinically and biochemically significant haemolysis, neutropenia, thrombocytopenia and AKI were used. RESULTS: 1919 infusion episodes in 90 patients were analysed. Age(mean(S.D)) = 58.09(14.4)years, 63% male, 72% CIDP(28% MMN), 97% IVIg(3% SCIg). Dose = 1.57(0.79)g/kg/month or 97.1(37.3)g/infusion, frequency:3.9(1.4) weeks. Relative IgA deficiency was noted in 2 individuals (prevalence:2.2%, 95%C.I.:0-5.2) who received a combined total of 38 infusions(3800 g IVIg) without adverse event. No clinically significant episodes of haemolysis, neutropenia, thrombocytopenia or AKI occurred in relation to treatment. An asymptomatic drop>10 g/L haemoglobin(Hb) occurred in 3.5%(95%CI:2.7-4.3) of treatment episodes in 38 individuals, mean reduction:17.7(7.4)g/L; lowest Hb:86 g/L. Lower pre-treatment haemoglobin correlated with risk of recurrent Ig-related drop(p:0.007). Two patients with chronic renal failure(stage 1 and 3) received 28(IV) and 104(SC) infusions respectively(6416 g) without impact on estimated glomerular filtration rate(eGFR). CONCLUSIONS: No clinically significant Ig-related episodes of haemolysis or AKI were identified in this representative cohort. This suggests that routine monitoring is not essential in long-term Ig use but should be considered when clinically indicated.

Loss of human beta-defensin 1, 2, and 3 expression in oral squamous cell carcinoma.

INTRODUCTION: Human beta-defensins (HBDs) are cationic, antimicrobial peptides produced by epithelial cells and involved in various aspects of the innate and acquired immune responses. They are expressed by oral tissues as constitutive and inducible genes. Recently, single nucleotide polymorphisms (SNPs) of beta-defensins have been correlated with increased susceptibility to certain diseases. Studies have reported altered expression of beta-defensins in cancers suggesting their involvement in carcinogenesis. The purpose of this study was to evaluate the regulation of HBD-1 (also published as DEFB1), HBD-2 (DEFB4) and HBD-3 (DEFB103A) (http://www.genenames.org/index.html) and HBD-1 SNPs in oral squamous cell carcinoma cell lines (OSCC) and healthy gingival keratinocytes. METHODS: beta-defensin expression was quantitatively assessed using real-time polymerase chain reactions in OSCC and control cell lines after exposure to interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma. Control data were obtained in a previous study. DNA from 19 OSCC cell lines and 44 control subjects were extracted and the HBD-1 region spanning the 5' untranslated region to the first intron was sequenced and analysed for SNP identification and distribution. RESULTS: HBD-1 and HBD-2 basal messenger RNA expression were significantly lower in OSCC. In addition, the ability to be induced was significantly reduced in OSCC for all three beta-defensins. Four HBD-1 SNPs were differentially distributed between cancer and control populations. Genotype distribution at the HBD-1 locus also suggested loss of heterozygosity in OSCC. CONCLUSIONS: The genetic variation observed in OSCC compared with that in control cell lines may account for differences in beta-defensin expression. These results suggest a putative role for beta-defensins in carcinogenesis and indicate that beta-defensins may be useful markers of OSCC.

Protection against simian immunodeficiency virus vaginal challenge by using Sabin poliovirus vectors.

Here we provide the first report of protection against a vaginal challenge with a highly virulent simian immunodeficiency virus (SIV) by using a vaccine vector. New poliovirus vectors based on Sabin 1 and 2 vaccine strain viruses were constructed, and these vectors were used to generate a series of new viruses containing SIV gag, pol, env, nef, and tat in overlapping fragments. Two cocktails of 20 transgenic polioviruses (SabRV1-SIV and SabRV2-SIV) were inoculated into seven cynomolgus macaques. All monkeys produced substantial anti-SIV serum and mucosal antibody responses. SIV-specific cytotoxic T-lymphocyte responses were detected in three of seven monkeys after vaccination. All 7 vaccinated macaques, as well as 12 control macaques, were challenged vaginally with pathogenic SIVmac251. Strikingly, four of the seven vaccinated animals exhibited substantial protection against the vaginal SIV challenge. All 12 control monkeys became SIV positive. In two of the seven SabRV-SIV-vaccinated monkeys we found no virological evidence of infection following challenge, indicating that these two monkeys were completely protected. Two additional SabRV-SIV-vaccinated monkeys exhibited a pronounced reduction in postacute viremia to <10(3) copies/ml, suggesting that the vaccine elicited an effective cellular immune response. Three of six control animals developed clinical AIDS by 48 weeks postchallenge. In contrast, all seven vaccinated monkeys remained healthy as judged by all clinical parameters. These results demonstrate the efficacy of SabRV as a potential human vaccine vector, and they show that the use of a vaccine vector cocktail expressing an array of defined antigenic sequences can be an effective vaccination strategy in an outbred population.

Pharmacokinetics, metabolism, and excretion of irinotecan (CPT-11) following I.V. infusion of [(14)C]CPT-11 in cancer patients.

This study determined the disposition of irinotecan hydrochloride trihydrate (CPT-11) after i.v. infusion of 125 mg/m(2) (100 microCi) [(14)C]CPT-11 in eight patients with solid tumors. Mean +/- S.D. recovery of radioactivity in urine and feces was 95.8 +/- 2.7% (range 92.2-100.3%, n = 7) of dose. Radioactivity in blood, plasma, urine, and feces was determined for at least 168 h after dosing. Fecal excretion accounted for 63.7 +/- 6.8 (range 54.2-74.9%, n = 7) of dose, whereas urinary excretion accounted for 32.1 +/- 6.9% (range 21.7-43.8%; n = 7) of dose. One patient with a biliary T-tube excreted 30.1% of dose in bile, 14.2% in feces, and 48.2% in urine. Quantitative radiometric HPLC revealed that CPT-11 was the major excretion product in urine, bile, and feces. Aminopentane carboxylic acid (APC) and SN-38 glucuronide (SN-38G) were the most significant metabolites in urine and bile, whereas SN-38 and NPC, a primary amine metabolite, were relatively minor excretion products. SN-38 and APC were the most significant metabolites in feces. The relatively higher amount of SN-38 in feces compared with bile is presumably due to hydrolysis of SN-38G to SN-38 by enteric bacterial beta-glucuronidases. There was close correspondence between quantitative fluorescence HPLC and mass balance findings. CPT-11 was the major circulating component in plasma (55% of the mean radiochemical area under the curve), and CPT-11, SN-38, SN-38G, and APC accounted for 93% of the mean radiochemical AUC. These results show that the parent drug and its three major metabolites account for virtually all CPT-11 disposition, with fecal excretion representing the major elimination pathway.

Isoform-specific effects of charged residues at borders of the M1-M2 loop of the Na,K-ATPase alpha subunit.

The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain. Via a largely undefined mechanism, the ouabain affinity of the Na,K-ATPase can be manipulated by mutating the residues at the borders of the first extracellular (M1-M2) loop of the alpha subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue, we compared the effects of two combinations of charged residues at the M1-M2 loop border, R113, D124 and D113,R124 (numbered according to the rat alpha1 subunit), on the ouabain sensitivity of the alpha1 and alpha2 isoforms. We report that ouabain sensitivity is dependent not only upon the identity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chimeric alpha subunits reveals that the effects of potassium are determined primarily by the interaction of the N-terminus and M1-M2 loop with the C-terminal third of the alpha subunit. M1-M2 loop border residues may, therefore, influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na,K-ATPase catalytic cycle which is competent to bind ouabain.

Collaborative assessment: exploring parental injury prevention strategies through bicycle helmet use.

While multiple studies have investigated bicycle helmet use, qualitative studies investigating parental strategies to promote their children's safety are rare. Thirty-four parents were interviewed to explore their injury prevention strategies. Findings suggest that the developmental stage of the child, the child's gender, and rural versus urban residence are all related to strategies parents use and their success in promoting bicycle safety. Peer pressure was the major deterrent, and negative "parent pressure" was also identified as problematic. Themes emerged that may support future injury prevention efforts with children, parents, and their communities and provide agencies information not previously captured quantitatively.

Behavioral treatment of autistic persons: a review of research from 1980 to the present.

Studies evaluating behavioral treatment of autism from 1980 to the present were reviewed. Studies included were published in journal articles and utilized behavioral methodology. A total of 251 studies were included in the review. Each study was analyzed for target behaviors and behavioral techniques implemented. Target behaviors were divided into categories, which included aberrant behaviors, social skills, language, daily living skills, and academic skills. Behavioral techniques were classified as positive, negative, extinction, or combined. Results were presented for each category. Recent trends in the treatment literature were also reviewed, and recommendations for future research were presented.

A randomized trial of minoxidil in chemotherapy-induced alopecia.

BACKGROUND: Hair loss is a side effect of many chemotherapeutic agents, and patients have even refused possibly palliative or lifesaving drugs because they could not accept temporary or prolonged baldness. Topical minoxidil has been shown to be effective for androgenetic alopecia and alopecia areata. OBJECTIVE: Our purpose was to investigate the value and safety of minoxidil in chemotherapy-induced hair loss. METHODS: Twenty-two women who were facing adjuvant chemotherapy after breast surgery were registered in a protocol that used a 2% minoxidil topical solution or a placebo in a randomized double-blind trial. RESULTS: There was a statistically significant difference (favoring minoxidil) in the interval from maximal hair loss to first regrowth. Thus the period of baldness was shortened (mean, 50.2 days) in the minoxidil group. CONCLUSION: Minoxidil decreased the duration of alopecia caused by chemotherapy. There were no significant side effects.

Atrial natriuretic peptide-like (ANP-LIR) and ANP prohormone immunoreactive astrocytes and neurons of human cerebral cortex.

Atrial natriuretic peptide (ANP) represents a family of related peptides originally isolated from cardiac atria that have potent natriuretic, diuretic, and vasorelaxant properties. ANP has previously been localized in neurons of the rat brain in regions subserving cardiovascular functions and fluid/electrolyte balance and has been localized in astroglia of the canine brain. To determine whether ANP is present in astrocytes of the human brain and to validate the canine model for future studies, human brain tissue was obtained from autopsy cases with no brain damage or neurological or vascular disease. Human brains were obtained less than 3 h postmortem, and anterior cingulate and striate cortices were dissected following perfusion or immersion fixation. Immunohistochemical processing utilized antibodies against the processed form of ANP (ANP IV, ANP104-128) and against rat proANP (amino terminus) and the avidin-biotin-peroxidase technique. Isolated, strongly ANP-immunoreactive protoplasmic astrocytes were observed in all layers of the cingulate and striate cortex gray matter. ANP-positive fibrous astrocytes were observed in the white matter. Additionally, distinctive immunopositive astrocytes were found both within and immediately subjacent to the glia limitans. Antibody against the prohormone stained only protoplasmic astrocytes and sublimitans astrocytes and processes. In addition to the astroglia, ANP was detected in scattered multipolar neurons in the cerebral gray matter. These results provide additional evidence for diversity of peptide localization in astrocytes and suggest roles for ANP in the local regulation of cerebral blood flow, blood-brain barrier permeability, or cerebrospinal fluid volume.

Antibodies against highly conserved sites in the epidermal growth factor receptor tyrosine kinase domain as probes for structure and function.

We generated anti-peptide antibodies against four highly conserved sequences in the kinase domain and against two nonconserved sequences surrounding autophosphorylation sites in the carboxyl-terminal domain of the epidermal growth factor receptor (EGFR). These antibodies were used to examine topology and function in catalysis of specific sequences. Two of the highly conserved sites, HRD (residues 811-818) and DFG (residues 827-838), appeared to participate in catalysis since alpha HRD and alpha DFG but not the other anti-peptide antibodies inhibited EGFR kinase activity. Examination of the topology of the six sites revealed that epitopes in all except the HRD site appeared to be exposed to antibody binding in the EGFR. The conditions that caused increased exposure of the HRD site to interaction with antibody included autophosphorylation, addition of the ionic detergent sodium dodecyl sulfate (SDS), and elevation in temperature from 4 to 34 degrees C.

Structural and functional characterization of Escherichia coli peptidyl-prolyl cis-trans isomerases.

Peptidyl-prolyl cis-trans isomerases (PPIases), enzymes that catalyze the cis-trans isomerization of peptide bonds to which proline contributes the nitrogen, were purified from Escherichia coli. In this organism, at least two PPIases are present. Both the cationic (periplasmic) and anionic (cytoplasmic) PPIases are inhibited by cyclosporin A with a Ki of 25-50 microM, a concentration 1000-fold higher than that required for eukaryotic PPIases. Although isoelectric focusing indicates that the two enzymes differ in isoelectric point by at least 4.0 pH units, the specific activities of the enzymes toward the tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-methyl-coumarylamide are equivalent. The activity of both enzymes for a series of substituted succinyl-Ala-Xaa-Pro-Phe-para-nitroanilide tetrapeptides suggests that the structure and function of the active site of the prokaryotic proteins is similar to that of eukaryotic cyclophilins. Both enzymes are capable of catalyzing the refolding of thermally denatured type III collagen. Antibodies against the periplasmic PPIase do not recognize the cytoplasmic enzyme, indicating significant differences in epitopes between the two forms. Circular dichroism spectroscopy indicates that the secondary structure of the cationic protein consists of 17% alpha-helix, 34% beta-sheet, 17% turns, 33% random coil and is very similar to human cytosolic PPIase.

Comparison and item analysis of the MESSY for autistic and normal children.

Seventeen autistic children were matched for age, race, and sex with 17 nonautistic children, and group differences in social skills were assessed. Appropriate social skills and levels of inappropriate assertiveness/impulsiveness were assessed and evaluated using the Matson Evaluation of Social Skills with Youngsters (MESSY). Significant differences in both the appropriate and inappropriate social behaviors displayed by the two groups were found. The implications of these results are discussed.

The relationship of the biophysical and biochemical characteristics of type VII collagen to the function of anchoring fibrils.

Type VII collagen is a major component of anchoring fibrils, which are 800-nm-long centrosymmetrically cross-banded fibrils that are believed to secure the attachment of certain epithelial basement membranes to the underlying stromal matrix. The ultrastructure of the anchoring fibrils is highly variable, suggesting that the fibrils are flexible. Flexibility measurements along the length of the triple-helical domain of type VII procollagen indicate that major flexible sites correlate well with known discontinuities in the (Gly-X-Y)n repeating sequence. Therefore, the helical disruptions may account for the tortuous shapes of anchoring fibrils observed ultrastructurally. The centrosymmetrical banding pattern observed for anchoring fibrils results from the unstaggered lateral packing of antiparallel type VII collagen dimers that form these structures. This antiparallel arrangement is specified by disulfide bonds formed at the margins of a 60-nm overlap of the amino termini. As long as these disulfide bonds remain intact, they protect the amino-terminal overlapping triple helices from collagenase digestion. This disulfide-bonded pair of triple helices is termed C-1. Large nonhelical domains (NC-1) extend from both ends of the anchoring fibrils and are believed to interact with the basement membrane or with anchoring plaques. Rotary shadowing of the NC-1 domains showed trident-like shapes, suggesting that a single alpha-chain contributed the structure of each arm and that the three arms were extended. Biochemical and biophysical analyses of NC-1 domains independently confirm these suggestions and imply that the arms of NC-1 domains are identical and individually capable of interactions with basement membrane components, potentially allowing trivalent interaction of type VII collagen with various macromolecules.

The conformation of T4 bacteriophage dihydrofolate reductase from circular dichroism.

The secondary and tertiary structure of T4 bacteriophage dihydrofolate reductase is investigated by vacuum ultraviolet circular dichroism (CD) spectroscopy and probability analysis of the primary amino acid sequence. The far ultraviolet CD spectrum of the enzyme in the range of 260-178 nm is analyzed by the generalized inverse and variable selection methods developed by our laboratory. Variable selection yields an average content of 26% alpha-helix, 21% antiparallel beta-sheet, 10% parallel beta-sheet, 20% beta-turns, and 32% "other" structures within the T4 protein. The characteristic peaks of the CD spectrum indicate that the enzyme has a lot of antiparallel beta-sheet, which is typical of the alpha + beta tertiary class of globular proteins. The secondary structure of the protein is also analyzed by using four statistical methods on the amino acid sequence. Although the secondary structures predicted by each individual statistical method vary to a considerable extent, the fractions of each structure jointly predicted by a majority of the methods are in excellent agreement with our CD analysis. The alternating arrangement for some segments of alpha-helix and beta-sheet predicted from primary structure to be within the enzyme is characteristic of proteins containing parallel beta-sheet. This supports our conclusion that the protein contains both parallel and antiparallel beta-sheet structures, but finding both types of beta-sheet also means that the protein may have the variation on alpha/beta tertiary structure recently found in EcoRI endonuclease and thymidylate synthase. These observations, in conjunction with other physical properties of the T4 reductase, suggest that the enzyme perhaps shares an evolution in common with the dihydrofolate reductases derived from type I R-plasmids rather than with the host-cell protein.

Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication.

Inverse circular dichroism (CD) spectra are presented for each of the five major secondary structures of proteins: alpha-helix, antiparallel and parallel beta-sheet, beta-turn, and other (random) structures. The fraction of the each secondary structure in a protein is predicted by forming the dot product of the corresponding inverse CD spectrum, expressed as a vector, with the CD spectrum of the protein digitized in the same way. We show how this method is based on the construction of the generalized inverse from the singular value decomposition of a set of CD spectra corresponding to proteins whose secondary structures are known from X-ray crystallography. These inverse spectra compute secondary structure directly from protein CD spectra without resorting to least-squares fitting and standard matrix inversion techniques. In addition, spectra corresponding to the individual secondary structures, analogous to the CD spectra of synthetic polypeptides, are generated from the five most significant CD eigenvectors.

Low expression of human histocompatibility leukocyte antigen-DR is associated with hypermethylation of human histocompatibility leukocyte antigen-DR alpha gene regions in B cells from patients with systemic lupus erythematosus.

The relationship between the expression of HLA-DR antigens and the HLA-DR alpha gene methylation was examined in systemic lupus erythematosus (SLE). Using permanent B cell lines, we found reduced DR expression in SLE. The low DR expression was correlated with high anti-DNA antibody titers in patients' sera. The amounts of DR alpha message were lower in SLE cells than in normal controls, suggesting that the low expression of DR antigens is associated with gene functions. The extent of DNA methylation was examined at five CCGG sites in the HLA-DR alpha locus. DNA from both SLE and normal cells showed variable methylation patterns. Since the DR alpha gene is a single-copy gene, such a variability is the result of assaying a mixture of transformed clones containing methylated DR alpha gene, with other clones containing unmethylated DR alpha gene. A distinctive feature of normal cells was a consistent methylation pattern: 12 normal cell lines showed exactly the same pattern. In contrast, 28 SLE cell lines showed a cell-line-specific methylation, and hypermethylation at the DR alpha locus. The hypermethylation is often associated with transcriptionally inactive genes. Thus, our results suggest that (a) B cells with hypermethylated DR genes might express no or few DR antigens; (b) the ratio of cells with differently methylated DR genes is consistent in normal individuals, while, in SLE patients, cells with hypermethylated DR genes predominate, resulting in apparently reduced DR antigen expression; and (c) the aberrant DR expression could be associated directly with immunoregulatory dysfunctions in SLE disease.

Nuclear DNA degradation in lymphocytes of patients with systemic lupus erythematosus.

The degradation of nuclear DNA in lymphocytes of patients with systemic lupus erythematosus (SLE) was analyzed. A low m.w. DNA fragment was identified in phytohemagglutinin (PHA)-stimulated lymphocytes from both healthy controls and SLE patients. The rate of appearance of low m.w. DNA was at least fourfold higher in lymphocytes from SLE patients, suggesting that DNA from SLE cells are more easily degraded than that from normal cells. PHA-stimulated lymphocytes from SLE patients were also shown to be more sensitive to UV irradiation than those of controls. These results suggest that DNA metabolism is abnormal in T cells of SLE patients.