The Crawford variant as a cause of RhD typing discrepancies in blood banks: A case report

We present the case of a 55-year-old Colombian male who showed a discrepancy in the serological typing of the RhD antigen in his first platelet donation. The discrepancy persisted after a serological investigation with multiple Anti-D monoclonal reagents (IgG and IgM) under different conditions (22 °C and 37 °C, saline, and LISS/Coombs). Furthermore, partial RhD typing was performed, obtaining negative results with a commercially available panel of six Anti-D reagents. Molecular analysis showed a homozygous deletion of RHD and heterozygosity for the Crawford variant (RHCE*ce, RHCE*ceCF), with a predicted phenotype of C–, c+, E–, e+, Vs+, V+. Following the investigation of this case, this man has made 14 platelet donations showing variable reactivity, with agglutinations ranging from – to 2+. Since Crawford red blood cells express some RhD antigen epitopes, they could cause alloimmunization in RhD negative receptors. Likewise, Anti-D alloantibodies have been documented in Crawford variant carriers. Therefore, it is recommended that carriers of this variant be classified as RhD positive if they are blood donors and RhD negative if they are transfusion recipients. Also, in pregnant women carrying a Crawford variant, Anti-D immunoprophylaxis is recommended.     

Estimation of the minimum number of test specimens for fatigue testing of acrylic bone cement

In the literature on fatigue testing of acrylic bone cements, data sets of various sizes have been used in different test series for the same cement formulation. There are two important consequences of this situation. First, it means that some test series last much longer than others, with all the implications for the cost of testing. Second, it makes drawing conclusions about the fatigue performance of a cement, based on the results of different literature series, a problematic issue. Clearly then, a recommendation as to what should be the minimum number of test specimens to use that would allow for confidence in the results of the statistical treatment of the test results (Gmin) would be desirable. In the present work, a method that could be used to culminate in such a recommendation is described. This method involves (i) obtaining experimental fatigue test results and (ii) analyzing those results using the Weibull probability distribution function and other statistical methods. This methodology is illustrated using fatigue life results obtained from uniaxial tension-compression fatigue tests on specimens fabricated from the polymerizing dough of one commercially available acrylic bone cement. For a tolerable error of 5%, we estimated Gmin to be either 7 (if the fatigue life results are treated using the two-parameter Weibull distribution function) or 11 (if the fatigue life results are treated using the three-parameter Weibull distribution function). To be on the conservative side, we therefore recommend that Gmin be 11. Three key limitations of the methodology presented here are discussed.     

Modelling of chronic wound healing dynamics

Following chronic wound area over time can give a general overview of wound healing dynamics. Decrease or increase in wound area over time has been modelled using either exponential or linear models, which are two-parameter mathematical models. In many cases of chronic wound healing, a delay of healing process was noticed. Such dynamics cannot be described solely with two parameters. The reported study deals with two-, three-, and four-parameter models. Assessment of the models was based on weekly measurements of 226 chronic wounds of various aetiologies. Several quantitative fitting criteria, i.e. goodness of fit, handling missing data and prediction capability, and qualitative criteria, i.e. number of parameters and their biophysical meaning were considered. The median of goodness of fit of three- and four-parameter models was between 0.937 and 0.958, and the median of two-parameter moels was 0.821 to 0.883. Two-parameter models fitted wound area over time significantly (p=0.001) worse than three- and four-parameter models. The criterion handling missing data provided similar results, with no significant difference between three- and four-parameter models. Median prediction error of two-parameter models was between 111 and 746; three-parameter models resulted in an error of 64 to 128, and finally four-parameter models resulted in the highest prediction error of 407 and 238. Based on the values of quantitative fitting criteria obtained, three parameters were chosen as the most appropriate. Based on qualitative criteria, the delayed exponential model was selected as the most general three-parameter model. It was found to have good prediction capability and in this capacity it could be used to help physicians choose the most appropriate treatment for patients with chronic wounds after an initial three-week observation period, when the median error increase of fitting is 74%.     

The dose--response relation for rat spinal cord paralysis analyzed in terms of the effective size of the functional subunit

Radiobiological models for estimating normal tissue complication probability (NTCP) are increasingly used in order to quantify or optimize the clinical outcome of radiation therapy. A good NTCP model should fulfill at least the following two requirements: (a) it should predict the sigmoid shape of the corresponding dose-response curve and (b) it should accurately describe the probability of a specified response for arbitrary non-uniform dose delivery for a given endpoint as accurately as possible, i.e. predict the volume dependence. In recent studies of the volume effect of a rat spinal cord after irradiation with narrow and broad proton beams the authors claim that none of the existing NTCP models is able to describe their results. Published experimental data have been used here to try to quantify the change in the effective dose (D(50)) causing 50% response for different field sizes. The present study was initiated to describe the induction of white matter necrosis in a rat spinal cord after irradiation with narrow proton beams in terms of the mean dose to the effective volume of the functional subunit (FSU). The physically delivered dose distribution was convolved with a function describing the effective size or, more accurately, the sensitivity distribution of the FSU to obtain the effective mean dose deposited in it. This procedure allows the determination of the mean D(50) value of the FSUs of a certain size which is of interest for example if the cell nucleus of the oligodendrocyte is the sensitive target. Using the least-squares method to compare the effective doses for different sizes of the functional subunits with the experimental data the best fit was obtained with a length of about 9 mm. For the non-uniform dose distributions an effective FSU length of 8 mm gave the optimal fit with the probit dose-response model. The method could also be used to interpret the so-called bath and shower experiments where the heterogeneous dose delivery was used in the convolution process. The assumption of an effective FSU size is consistent with most of the effects seen when different portions of the rat spinal cord are irradiated to different doses. The effective FSU length from these experiments is about 8.5 +/- 0.5 mm. This length could be interpreted as an effective size of the functional subunits in a rat spinal cord, where multiple myelin sheaths are connected by a single oligodendrocyte and repair is limited by the range of oligodendrocyte progenitor cell diffusion. It was even possible to suggest a more likely than uniform effective FSU sensitivity distribution from the experimental data.     

The distribution of quick phase interval durations in human optokinetic nystagmus

There is an interesting dichotomy between models that predict the quick phase interval durations (QPIDs) of human optokinetic nystagmus (OKN). Accumulator models describe a stochastic signal in a neural network that triggers a response once the signal reaches a fixed threshold value. However, it is also possible that quick phases are triggered after eye position reaches a variable amplitude threshold. In this study, we fitted a range of probability density functions previously predicted by stochastic models of OKN (including those of the reciprocal truncated Normal, inverse Gaussian, gamma, lognormal and the mixture of two reciprocal truncated Normal distributions) to individual QPID histograms. We compared the goodness of fit between these models, and a model where the distribution of QPIDs is determined by the ratio of two correlated and truncated Normal random variables. The ratio distribution gave the best fit to the data, and we propose this is due to the approximately linear trajectory of slow phases (SPs) and that QPIDs are given by the ratio of a variable SP amplitude threshold and variable SP velocity.     

Carnitine palmitoyltransferase 1A (CPT1A) P479L prevalence in live newborns in Yukon,Northwest Territories,and Nunavut

Carnitine palmitoyltransferase 1A (CPT1A), encoded by the gene CPT1A, is the hepatic isoform of CPT1 and is a major regulatory point in long-chain fatty acid oxidation. CPT1A deficiency confers risk for hypoketotic hypoglycaemia, hepatic encephalopathy, seizures, and sudden unexpected death in infancy (SUDI). It remains controversial whether the CPT1A gene variant, c.1436C>T (p.P479L), identified in Inuit, First Nations, and Alaska Native infants, causes susceptibility to decompensation, in particular during times of fever and intercurrent illness. Although newborn screening for the P479L variant occurs in some jurisdictions, background knowledge about the presence of the variant in Canadian Aboriginal populations is lacking. In an effort to understand the population implications of the variant in northern Canada, overall frequencies of the variant were assessed. Further studies are underway to determine associated risk. Ethics approval was obtained from university REBs, local research institutes, and with consultation with territorial Aboriginal groups. Newborn screening blood spots from all infants born in 2006 in the three territories were genotyped for the p.P479L variant. p.P479L (c.1436C>T) allele frequencies in the three territories were 0.02, 0.08, and 0.77 in Yukon (n = 325), Northwest Territories (n = 564), and Nunavut (n = 695), respectively. Homozygosity rates were 0%, 3%, and 64%. Aboriginal status was available only in NWT, with allele frequencies of 0.04, 0.44, 0.00, and 0.01 for First Nations, Inuvialuit/Inuit, Métis, and non-Aboriginal populations. Although individual blood spots were not identified for Aboriginal ethnicity in Nunavut infants, ~ 90% of infants in Nunavut are born to Inuit women. The allele frequency and rate of homozygosity for the CPT1A P479L variant were high in Inuit and Inuvialuit who reside in northern coastal regions. The variant is present at a low frequency in First Nations populations, who reside in areas less coastal than the Inuit or Inuvialuit in the two western territories. The significance of the population and geographic distribution remains unclear, but the high population frequencies of the variant suggest a historically low penetrance for adverse outcomes. Further evidence is needed to determine if there is an increased risk for infant mortality and morbidity and whether newborn screening will be indicated on a population basis.     

Choice of Ascertainment Model II

Probability models have been developed for family data, where each family has been selected or ascertained through a group of m probands, where m is a known parameter, not a random variable. The birth orders of the probands among the affected children are reported for each family. It is demonstrated that this information is sufficient for choice of ascertainment model. The conditional probability that an ascertained family with s children has r affected ones, depends, in addition to the segregation parameter, on the birth order of the youngest proband only. By means of the joint distribution of the birth orders of the other probands, it can be demonstrated if the ascertainment takes place through affected children near to or distant from each other in relative birth orders. Statistical methods have been developed for cases with two and cases with m probands.     

Assessment of gait sensitivity norm as a predictor of risk of falling during walking in a neuromusculoskeletal model

Quantifying the risk of falling (falls risk) would be helpful in treating people with gait disorders. The gait sensitivity norm (GSN) is a stability measure that correlates well to risk of falling in passive dynamic walkers but has not been evaluated on humans or human-like walking models. We assessed the correlation of GSN to risk of falling in a neuromusculoskeletal (NMS) walking model. Specifically, we evaluated the correlation of GSN to the actual disturbance rejection (ADR) of the model and the sensitivity of this relationship to gait parameter, Poincaré section selection and steady state variability correction. Statistically significant results at p < 0.05 were obtained for some of the gait indicators evaluated at the point in the gait cycle where they were most variable. The correlation between GSN and ADR was sensitive to gait indicator and Poincaré sections evaluated but not to steady state variability correction. The current work suggests some simple steps to reduce the sensitivity of GSN to arbitrary and subjective factors. Overall, the findings support the potential of GSN to be a clinically applicable measure of falls risk. Further study is required to identify methods to more definitively select the various factors within the GSN calculation and to confirm its ability to predict falls risk in human subjects.     

Thermodynamic determination of beta-hexosaminidase isoenzymes in mononuclear and polymorphonuclear leukocyte populations

Isoenzymes of beta-hexosaminidase (Hex) were determined in mononuclear (MN) and polymorphonuclear (PMN) leukocytes, with a thermodynamic method using the chromogenic substrate sodio-3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide. Imprecision was very satisfactory, and the results are very much in agreement with those obtained using the fluorogenic substrates 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide 6-sulfate. In 163 healthy individuals we found, for the proportion as a percentage of the Hex A isoenzyme, significantly higher values (P < 0.001) in PMN than in MN cells (71.56 +/- 0.30% vs. 54.28 +/- 0.24%), meaning that it would not appear advisable to use total leukocyte lysates for evaluating this variable. The method is fast, precise, and highly suitable for the biochemical diagnosis and heterozygote screening of GM2 gangliosidoses, and would be applicable in cases of thermolabile Hex B and for detecting the B1 variant.     

Semiquantitative and qualitative assessment of B-lymphocyte V H repertoire by a fluorescent multiplex PCR

We established a new tool to perform semiquantitative and qualitative screening for V(H) gene usage frequency during IgH rearrangements in human B-lymphocytes. In two separate multiplex PCRs, the rearranged VDJ regions were amplified with V(H) family-specific primers labeled with different fluorescent dyes (FAM, HEX, NED, or ROX). The relative amount of each of the particular V(H) family products and their ratios were determined by fragment analysis on a ABI PRISM 377 sequencer. We verified that the fluorescent multiplex PCR (FMPCR) shows high specificity and sensitivity, acceptable reproducibility and reliability. Data obtained were well in agreement with results revealed by sequencing following single-cell PCR. Ten healthy volunteers showed a comparable semiquantitative V(H) family distribution. The FMPCR also correctly detected a monoclonal peak in a CLL patient. Thus, labeling primers with various fluorescent dyes allows for an assessment of V(H) family usage and an immediate determination of the involved V(H) gene family if any clonal peaks are present. This method provides a quick, easy, and reliable tool for V(H) repertoire screening of larger populations of patients suffering from diseases with changes in the V(H) repertoire allowing for selection of cases worth a more detailed and cumbersome sequence analysis later on.     

Divergent biophysical properties, gating mechanisms, and possible functions of the two skeletal muscle CaV1.1 calcium channel splice variants

Voltage-gated calcium channels are multi-subunit protein complexes that specifically allow calcium ions to enter the cell in response to membrane depolarization. But, for many years it seemed that the skeletal muscle calcium channel Ca(V)1.1 is the exception. The classical splice variant Ca(V)1.1a activates slowly, has a very small current amplitude and poor voltage sensitivity. In fact adult muscle fibers work perfectly well even in the absence of calcium influx. Recently a new splice variant of the skeletal muscle calcium channel Ca(V)1.1e has been characterized. The lack of the 19 amino acid exon 29 in this splice variant results in a rapidly activating calcium channel with high current amplitude and good voltage sensitivity. Ca(V)1.1e is the dominant channel in embryonic muscle, where the expression of this high calcium-conducting Ca(V)1.1 isoform readily explains developmental processes depending on L-type calcium currents. Moreover, the availability of these two structurally similar but functionally distinct channel variants facilitates the analysis of the molecular mechanisms underlying the unique current properties of the classical Ca(V)1.1a channel.     

Estimation of the electrical parameters of spinal motoneurons using impedance measurements

Electrical parameters of spinal motoneurons were estimated by optimizing the parameters of motoneuron models to match experimentally determined impedance functions with those of the models. The model was described by soma area, somatic and dendritic membrane resistivities, and the diameter of an equivalent dendritic cable having a standard profile. The impedance functions of motoneurons and optimized models usually differed (rms error) by <2% of input resistance. Consistent estimates for most parameters were obtained from repeated impedance determinations in individual motoneurons; estimates of dendritic resistivity were most variable. The few cells that could not be fit well had reduced impedance phase lag consistent with dendritic penetrations. Most fits were improved by inclusion of a voltage-dependent conductance G(V) with time constant tau(V). A uniformly distributed G(V) with tau(V) >5 ms provided a better fit for most cells. The magnitude of this conductance decreased with depolarization. Impedance functions of other cells were adequately fit by a passive model or by a model with a somatic G(V) and tau(V) <5 ms. Most of these neurons (7/8) had resting potentials positive to -60 mV. The electrotonic parameters rho, tau, and L, estimated from model parameters, were consistent with published distributions. Most motoneuron parameters obtained in somatic shunt and sigmoidal models were well correlated, and parameters were moderately affected by changes in dendritic profile. These results demonstrate the utility and limitations of impedance measurements for estimating motoneuron parameters and suggest that voltage-dependent conductances are a substantial component of resting electrical properties.     

Next Generation Sequencing Identifies a Novel Rearrangement in the HBB Cluster Permitting to‐the‐Base Characterization

Genetic testing for hemoglobinopathies is required for prenatal diagnosis, understanding complex cases where multiple pathogenic variants may be present or investigating cases of unexplained anemia. Characterization of disease causing variants that range from single base changes to large rearrangements may require several different labor‐intensive methodologies. Multiplex ligation probe amplification analysis is the current method used to detect indels, but the technique does not characterize the breakpoints or detect balanced translocations. Here, we describe a next‐generation sequencing (NGS) method that is able to identify and characterize a novel rearrangement of the HBB cluster responsible for εγδβ thalassemia in an English family. The structural variant involved a 59.0 kb inversion encompassing HBG2 exon 3, HBG1, HBD, HBB, and OR51V1, juxtaposed by a deletion of 122.6 kb including 82 bp of the inverted sequence, HBG2 exon 1 and 2, HBE, and the β‐locus control region. Identification of reads spanning the breakpoints provided to‐the‐base resolution of the rearrangement, subsequently confirmed by gap‐PCR and Sanger sequence analysis. The same rearrangement, termed Inv‐Del English V εγδβ thalassemia (HbVar 2935), was identified in two other unrelated English individuals with a similar hematological phenotype. Our NGS approach should be applicable as a diagnostic tool for other disorders.     

Aberrant expression of immunoglobulin heavy chain genes in Epstein-Barr virus-negative, human immunodeficiency virus-related lymphoid interstitial pneumonia

The two-step polymerase chain reaction (PCR) and sequencing analysis was used to analyze the immunoglobulin heavy chain variable (Ig V(H)) genes of open-chest biopsy or autopsy samples from five patients with Epstein-Barr virus-negative human immunodeficiency virus (HIV)-related lymphoid interstitial pneumonia (LIP), and the results were compared with those for Ig V(H) genes from five HIV-negative LIP patients. The findings of this study are consistent with the different immunological situations of HIV-related and HIV-negative LIP. (a) The Ig V(H)3 subgroup was underexpressed in three of five cases of HIV-related LIP. In contrast, none of the HIV-negative cases showed this abnormality. Because the Ig V(H)3 subgroup encodes the largest portion of Ig V(H) genes, a depletion of B cells expressing Ig V(H)3 genes reflects a major alteration in the B-cell compartment. (b) All HIV-related LIP cases demonstrated two or three oligoclonal populations. HIV-negative cases showed minor monoclonal or polyclonal populations, but not oligoclonal ones. These oligoclonal populations suggest the coexistence of several occult clonal B-cell populations in HIV-related LIP. (c) Some oligoclonal clones in HIV-related LIP showed mutated framework regions not demonstrated in HIV-negative clones. This degree of variation exceeds the usual mutation rate for frameworks, suggesting a role for framework residues in antigen binding. (d) The frequency of D-D fusions of minor oligoclonal clones (HIV-related LIP) is higher than that of minor monoclonal clones (HIV-negative LIP). Such D-D fusions may enhance the probability of expression of antibodies capable of binding HIV glycoproteins.     

DNA sequence analysis of GJB2, encoding connexin 26: observations from a population of hearing impaired cases and variable carrier rates, complex genotypes, and ethnic stratification of alleles among controls

Mutations in GJB2 are associated with hereditary hearing loss. DNA sequencing of GJB2 in a cohort of hearing impaired patients and a multi-ethnic control group is reported. Among 610 hearing impaired cases, 43 DNA sequence variations were identified in the coding region of GJB2 including 24 mutations, 8 polymorphisms, 3 unclassified variants (G4D, R127C, M163V), 1 controversial variant (V37I), and 7 novel variants (G12C, N14D, V63A, T86M, L132V, D159, 592_600delinsCAGTGTTCATGACATTC). Sixteen non-coding sequence variations were also identified among cases including the IVS1+1A>G mutation, 2 polymorphisms, and 13 novel variants. A diagnosis of GJB2-associated hearing loss was confirmed for 63 cases (10.3%). Heterozygous mutations were found in 39 cases (6.4%). Eleven cases carrying novel or unclassified variants (1.8 %) and 18 cases carrying the controversial V37I variant were identified (3%). In addition, 294 control subjects from 4 ethnic groups were sequenced for GJB2. Thirteen sequence variations in the coding region of GJB2 were identified among controls including 2 mutations, 6 polymorphisms, 2 unclassified variants (G4D, T123N), 1 controversial variant (V37I), and 2 novel variants (R127L, V207L). Nine sequence variations were identified among controls in the non-coding regions in and around GJB2 exon 2. Of particular interest among controls were the variability in carrier rates and ethnic stratification of alleles, and the complex genotypes among Asians, 47% of whom carried two to four sequence variations in the coding region of GJB2. These data provide new information about carrier rates for GJB2-based hearing loss in various ethnic groups and contribute to evaluation of the pathogenicity of the controversial V37I variant.     

Immunohistochemical Features of Giant Cell Carcinoma of the Lung: Patterns of Expression of Cytokeratins, Vimentin, and the Mucinous Glycoprotein Recognized by Monoclonal Antibody A-80

Giant cell carcinoma of the lung (GCCL) is an uncommon and extremely aggressive variant of lung cancer. Characteristic microscopic findings include marked pleomorphism, aggregates of mononucleated or multinucleated giant cells (or both), a general lack of architectural cohesiveness, extensive necrosis, and endocytosis by the giant cells. Although the epithelial character of GCCL has been confirmed by a number of studies, controversy persists as to whether it represents a variant of poorly differentiated adenocarcinoma or of squamous carcinoma. Histochemical studies for mucosubstances have yielded variable and conflicting results. This report describes conventionally fixed and processed samples from 10 cases of GCCL studied with a panel of monoclonal antibodies (Mabs) recognizing different cytokeratin polypeptides (AE1, AE3, AE1/AE3 cocktail, and CAM 5.2), vimentin, and Mab A-80, the last of which binds to a mucinous glycoprotein associated with exocrine differentiation. All 10 cases of GCCL reacted with all cytokeratin Mabs; the extent and intensity of the reaction varied notably. All cases stained strongly and diffusely with Mab AE1 and AE1/AE3, the reaction was less extensive and weaker with CAM 5.2. Significantly, 2 cases reacted focally with Mab AE3. Nine cases reacted extensively and intensely with the vimentin Mab, often showing prominent paranuclear globular profiles. All cases reacted with Mab A-80; the reaction was often strong, but the extent was variable. Findings indicate that all GCCL are indeed cytokeratin positive but that most express polypeptides toward the low-molecular weight end of the spectrum; a small subset also expresses heavier polypeptides. This profile suggests that all GCCL display cytokeratins characteristic of adenocarcinoma but that a subset also shows polypeptides typical of squamous carcinomas; significantly, all GCCL retained their exocrine phenotype as defined by their positivity with Mab A-80. Moreover, the consistent if not invariable finding of vimentin positivity adds to an immunohistochemical profile that is distinct from that of the vast majority of lung cancers.     

单胺氧化酶A基因EcoRⅤ多态与帕金森病的关系分析

目的 研究单胺氧化酶A（monoamine oxidase A,MAO-A）基因EcoRⅤ多态（C／T）位点在中国人群中的分布，并探讨其与帕金森病（Parkinson's disease,PD)发病风险的关系。方法 采用聚合酶链反应－限制性片段长度多态性分析法，在110例PD患者和182名正常人中分析了MAO－A基因EcoRⅤ（C／T）多态的分布，并对该多态与PD进行关联分析。结果 （1）MAO－A基因EcoRⅤ多态与PD间不存在明显关联（x^2=0.091,P=76.3）；（2）MAO－A基因EcoRⅤ多态在中国汉族人群和北美高加索人群中的分布差异有显著性（x^2＝30.03,P=4.18）。结论 MAO－A EcoRⅤ多态有与中国人PD的发病风险无关。     

A simple estimate of the general population frequency of the MHC susceptibility gene for autoimmune polygenic disease

We wished to determine the frequencies of the MHC and non-MHC susceptibility genes for polygenic autoimmune diseases like type 1 diabetes (IDDM). We used Mendelian inheritance and the Hardy-Weinberg equilibrium to calculate the frequencies of mating pairs and susceptible offspring under classical recessive and dominant inheritance of the MHC susceptibility gene. We then analyzed the distribution of haplotype sharing by affected sib pairs of the 4 MHC haplotypes in each of the kinds of mating pairs in terms of the frequency of the disease susceptibility gene. For IDDM, the analysis was consistent with a recessive, but not a dominant, MHC susceptibility gene of frequency 0.525 at a distribution of 55, 38 and 7% of affected sib pairs who share 2, 1 and 0 MHC haplotypes, respectively. A simple relationship was obtained: if inheritance is recessive, the MHC susceptibility gene frequency is the square root of the fraction of affected sib pairs who share no MHC haplotypes multiplied by 4. For recessive inheritance, affected sib pairs who share no haplotypes are solely in families where both parents are homozygous MHC-susceptible. Although homozygous MHC susceptibles represent over 25% of the population, only 2-3% of them are IDDM-susceptible at non-MHC susceptibility loci, also required for disease expression. Predictions from our analysis fit all published observations of the familial occurrence of disease. The analysis is general, simple and provides a single estimate (not a range) of the MHC susceptibility gene frequency. This approach should be applicable to other MHC-determined polygenic diseases.     

Properties of a conductive cellular chloride pathway in the skin of the toad (Bufo bufo)

Two types of chloride current response to a step-wise hyperpolarization of the toad skin is demonstrated: (1) An “instantaneous” response observed immediately upon voltage change, and (2) a subsequent slow response, the time course of which is sigmoidal. The slow response is due to an increase of a transcellular conductance which is specific to chloride ions. The time constant of the conductance increase is dependent on the amplitude of the transepithelial voltage displacement, the smallest time constants are obtained for the highest amplitudes and are in the order of 30 s. The voltage dependences of the steady-state conductance and the steady-state chloride current reveal that the chloride pathway has maximum conductance for V?-80 mV (outside of the skin being negative) and approaches a non-conducting state for V > 0 mV. This strong outward going rectification is a steady-state phenomenon: In skins hyperpolarized for a few minutes, the “instantaneous” I-V curves show that the chloride pathway in the conducting state allows a large inward chloride current (outward chloride flux) to pass in the voltage range 40 mV > V > 0 mV. Calculations based on a three-compartment model indicate that the strong steady-state chloride current rectification cannot be obtained if only the intracellular chloride concentration and the membrane potentials are allowed to vary (“Goldman-rectification”). It is suggested, therefore, that the permeability of the chloride pathway varies reversibly with the transepithelial potential difference. The variable which controls the chloride permeability may be a membrane potential or the concentration of an intracellular ion.