A comparative study of the traditional method, and a point-score system for allocation of deceased-donor kidneys: a national multicenter study in Mexico.

BACKGROUND: The National Transplant Center in Mexico has ruled that deceased-donor kidney allocation is a function of each hospital's Internal Transplant Committee. The aim of this study was to compare and analyze results for of the traditional method and a point-score system in the allocation of deceased patient's kidneys. METHODS: The 12 major kidney transplant centers in the country having a deceased-donor program were invited to participate. Only 3 of them replied to the invitation during 2010. A point-score system was proposed to them, comprising blood group, waiting list time, HLA type, and donor and recipient ages. Once the final recipient was chosen, an explanation of reasons for the choice was requested. Thirty-eight transplants were presented. Kappa coefficient was used to measure degree of agreement in both allocation systems. Organs donated for transplantation came from patients between 4 and 54 years old, including 52% female, 52% O+ blood type, 31% A+, and 11% B+, 44% cranial-encephalic trauma, and 44% brain hemorrhage. RESULTS: Global agreement was 52.6% (kappa = 0.343), and partial agreement was 76.3% (weighted kappa = 0.204), assigning more intensity to extreme values, but with a lower correlation index. A more intense agreement, without discriminating by hospital, was found for "A" category (blood group), followed by "B" category (waiting list time). DISCUSSION: Taking into consideration the determining factors for long-term graft survival, it is indispensable to include criteria such as donor and recipient ages and HLA typife in the allocation process. This first draft of a point-score system in organ allocation included waiting list time, blood group, urgency related to vascular/peritoneal access for dialysis, clinical condition, donor/recipient age ratio, and HLA antigenic compatibility.

Role of 5-HT2A, 5-HT4 and 5-HT7 receptors in the antigen-induced airway hyperresponsiveness in guinea-pigs.

BACKGROUND: A possible role of 5-hydroxytryptamine (5-HT) in the origin of antigen-induced airway hyperresponsiveness (AI-AHR) has been scarcely investigated. OBJECTIVE: To explore the participation of different 5-HT receptors in the development of AI-AHR in guinea-pigs. METHODS: Lung resistance was measured in anaesthetized guinea-pigs sensitized to ovalbumin (OVA). Dose-response curves to intravenous (i.v.) acetylcholine (ACh) were performed before and 1 h after antigenic challenge and expressed as the 200% provocative dose (PD(200)). Organ bath experiments, confocal microscopy and RT-PCR were additionally used. The 5-HT content in lung homogenates was measured by HPLC. RESULTS: Antigenic challenge significantly decreased PD(200), indicating the development of AI-AHR. This hyperresponsiveness was abolished by a combination of methiothepin (5-HT(1)/5-HT(2)/5-HT(5)/5-HT(6)/5-HT(7) receptors antagonist) and tropisetron (5-HT(3)/5-HT(4) antagonist). Other 5-HT receptor antagonists showed three different patterns of response. Firstly, WAY100135 (5-HT(1A) antagonist) and ondansetron (5-HT(3) antagonist) did not modify the AI-AHR. Secondly, SB269970 (5-HT(7) antagonist), GR113808 (5-HT(4) antagonist), tropisetron or methiothepin abolished the AI-AHR. Thirdly, ketanserin (5-HT(2A) antagonist) produced airway hyporresponsiveness. Animals with bilateral vagotomy did not develop AI-AHR. Experiments in tracheal rings showed that pre-incubation with LP44 or cisapride (agonists of 5-HT(7) and 5-HT(4) receptors, respectively) induced a significant increase of the cholinergic contractile response to the electrical field stimulation. In sensitized lung parenchyma strips, ketanserin diminished the contractile responses to ACh. Sensitization was associated with a ninefold increase in the 5-HT content of lung homogenates. Confocal microscopy showed that sensitization enhanced the immunolabelling and co-localization of nicotinic receptor and 5-HT in airway epithelium, probably located in pulmonary neuroendocrine cells (PNECs). RT-PCR demonstrated that neither sensitization nor antigen challenge modified the 5-HT(2A) receptor mRNA levels. CONCLUSIONS: Our results suggested that 5-HT was involved in the development of AI-AHR to ACh in guinea-pigs. Specifically, 5-HT(2A), 5-HT(4) and 5-HT(7) receptors seem to be particularly involved in this phenomenon. Participation of 5-HT might probably be favoured by the enhancement of the PNECs 5-HT content observed after sensitization.

Permeant anions control gating of calcium-dependent chloride channels.

The effects of external anions (SCN(-), NO3-, I(-), Br(-), F(-), glutamate, and aspartate) on gating of Ca(2+)-dependent Cl(-) channels from rat parotid acinar cells were studied using the whole-cell configuration of the patch-clamp technique. Shifts in the reversal potential of the current induced by replacement of external Cl(-) with foreign anions, gave the following selectivity sequence based on permeability ratios ( P(x)/ P(Cl)): SCN(-)>I(-)>NO3->Br(-)>Cl(-)>F(-)>aspartate>glutamate. Using a continuum electrostatic model we calculated that this lyotropic sequence resulted from the interaction between anions and a polarizable tunnel with an effective dielectric constant of approximately 23. Our data revealed that anions with P(x)/P(Cl) > 1 accelerated activation kinetics in a voltage-independent manner and slowed deactivation kinetics. Moreover, permeant anions enhanced whole-cell conductance ( g, an index of the apparent open probability) in a voltage-dependent manner, and shifted leftward the membrane potential- g curves. All of these effects were produced by the anions with an effectiveness that followed the selectivity sequence. To explain the effects of permeant anions on activation kinetics and g(Cl) we propose that there are 2 different anion-binding sites in the channel. One site is located outside the electrical field and controls channel activation kinetics, while a second site is located within the pore and controls whole-cell conductance. Thus, interactions of permeant anions with these two sites hinder the closing mechanism and stabilize the channel in the open state.

Cellular immune response to Klebsiella pneumoniae antigens in patients with HLA-B27+ ankylosing spondylitis.

OBJECTIVE: To study the reactivity of peripheral blood mononuclear cells (PBMC) of patients with ankylosing spondylitis (AS) and rheumatoid arthritis (RA) and healthy controls to Klebsiella pneumoniae antigens and to the GroEL-like proteins from K. pneumoniae (HSP60Kp) and Mycobacterium leprae recombinant heat shock protein 65 (rHSP65Ml). METHODS: PBMC of 13 patients with AS and 9 with RA and 10 controls were stimulated in vitro by heat shock induced K. pneumoniae antigens in a cell blot assay, by insolubilized HSP60Kp, by cytosolic proteins (CP) from K. pneumoniae cultivated at 37 degrees C or 45 degrees C, by soluble HSP60Kp, or by rHSP65Ml. RESULTS: In the cell blot assay 7/13 AS and 3/9 RA patients responded to fraction 4, which contains mainly HSP60Kp, and no controls responded (AS vs. controls: p = 0.007). The response to the insolubilized HSP60Kp was positive in 6/13 AS patients but negative in RA patients and controls (p = 0.004). The response to CP45 degrees C was positive in 7/13 AS, in 2/9 RA, and no controls (AS vs controls: p<0.015). Response to the soluble HSP60Kp was found in 7/13 AS and 5/9 RA patients, but no controls (AS vs. controls: p = 0.0075). Response to rHSP65Ml was positive in 3/13 AS, 7/9 RA patients, and 1/10 controls (AS vs RA: p = 0.027; RA vs. controls: p = 0.005; AS vs. controls: nonsignificant). CONCLUSION: In PBMC of the majority of patients with AS and in some with RA, but not in healthy controls, there are cells that proliferate in the presence of HSP60 of K. pneumoniae.

Recognition of B cells epitopes of the Klebsiella pneumoniae GroEL-like protein by HLA-B27 positive subjects.

The presence of antibodies against antigens of K. pneumoniae in HLA-B27 positive patients with ankylosing spondylitis (AS), has been well documented. We have previously reported that sera from HLA-B27 positive subjects react with the K. pneumoniae GroEL-like protein (HSP60Kp) and have higher titers than HLA-B27 negative individuals. We cloned the gene that codes for this protein, determined hydrophilic regions by computer analysis of the predicted amino acid sequence and found that residues 389-397, 360-368 and 282-290, were possible B cell epitopes. To test this prediction, and to determine if the HLA-B27 positive and negative AS patients recognize the same or different epitopes, we truncated the hsp60Kp gene, from the 3; terminal nucleotide, to obtain fragments having or not the predicted epitopes. Four polypeptides of 40, 37, 30 and 18 kDa were obtained and analysed, by ELISA and inhibition of ELISA, for their reactivity with IgG antibodies from three high responders HLA-B27 positive AS patients and three HLA-B27 negative subjects who recognized the rHSP60Kp. Sera from both HLA-B27 positive and negative subjects reacted equally well with rHSP60Kp or with the 40 and 37 kDa peptides, which do not have residues 389-397 and 360-368, respectively, but reactivity was lost with the 30 kDa peptide, which also lacks residues 282-290. Contrary to what we expected, antibodies from HLA-B27 negative and positive individuals recognized the same epitope of the HSP60Kp. Our results indicate that the important epitope for B cells could be the 282-290 region and that the contribution of the two other predicted regions is minimal. We also conclude that the differences in response to the HSP60Kp in HLA-B27 positive AS patients and HLA-B27 negative individuals is not qualitative, but only quantitative.

Identification of peptides presented by HLA class I molecules on cervical cancer cells with HPV-18 infection.

In this work we eluted peptides from purified class I MHC molecules, isolated from a novel human cervical carcinoma cell line (INBL), generated in our laboratory and positive for HPV-18 infection. A fraction of these peptides was capable of stimulating T lymphocytes obtained from a donor matched for HLA-Cw4 and who was also HPV-18+. Direct N-terminal Edman degradation of these peptides, revealed the sequence (XQFPIFLQF) that matched 85% with the sequence NVFPIFLQM localized in between the 54 and 62 residues of the HPV-18 L1 protein. After stimulation with the synthetic peptide NVFPIFLQM, T lymphocytes from the donor were capable to lyse INBL cells. Our results provide evidence of the existence of naturally occurring viral epitopes presented on cervical cancer cells by the HLA-Cw4 allele, that could be useful for immunotherapy on this type of patient.

Actividad de superóxido dismutasa y las concentraciones de glutation y malonaldehído en los tejidos pulmonar y cardiaco post (perfusión, preservación y reperfusión) del bloque cardiopulmonar / Superoxide dismutase activity and glutathione and malonaldehyde concentration in pulmonary and cardiac tissue post (perfusion, preservation and reperfusion) of cardiopulmonary block

Introducción. Los periodos de isquemia y de reperfusión involucra la producción de radicales libres (RL), un grupo de especies oxidantes derivadas del matabolismo de oxígeno molecular. Durante la reperfusión, se generan (RL) al restablecer la circulación sanguínea dado el aporte de oxígeno, además de que durante la isquemia, el metabolismo aeróbico del bloque cardiopulmonar continúa por el oxígeno remanente en los alvéolos. La degradación de los RL es a través de receptores específicos como la enzima superóxido dismutasa (SOD) y el glutatión (GLU). Si estos mecanismos receptores no funcionan adecuadamente, los RL pueden dar inicio a la peroxidación lipídica. El manoaldehído (MAL) es un producto final de la peroxidación lipídica y su cuantificación refleja el daño celular por RL. Objetivo. Utilizando un modelo experimental murino de perfusión, preservación y reperfusión cardiopulmonar a través de la arteria pulmonar con solución de Krebs-Henseleit a 4ºC y con un flujo de 0.2 mL/min, el objetivo de este trabajo fue: determinar y evaluar la actividad de la enzima SOD y las concentraciones de GLU y MAL en homogeneizados tisulares de corazón y de pulmón como respuesta al daño ocasionado por efecto de la perfusión, preservación y reperfusión cardiopulmonar. Material y métodos. Utilizamos 14 bloques cardiopulmonares de murinos Balb-C divididos en dos grupos de estudio. Los bloques fueron: Grupo 1 (n=7): perfundidos en forma inmediata y continua únicamente durante el tiempo necesario para exaguinarlos y Grupo 2 (n=7): perfundidos en forma inmediata y continua para exanguinarlos, preservados durante 24 horas a 4ºC y reperfundidos durante 30' bajo las mismas condiciones dadas para la perfusión. Concluidas las perfusiones y la reperfusiones, preparamos homogeneizados del corazón y de los pulmones para determinar la actividad SOD y las concentraciones de GLU y MAL mediante espectrofotometría. Resultados. La actividad de SOD antes y después de la preservación fue mayor en el tejido pulmonar que en el tejido cardiaco. En ambos tejidos, la actividad de SOD y la concentración de GLU obtenidas previa preservación prolongada. La concentración de MAL fue similar en ambos homogeneizados tisulares tanto antes como después de la preservación del bloque cardiopulmonar y no se modificó por efecto de la reperfusión

Interleucinas 1ß, 6 y 10 y el daño tisular post-perfusión y post-reperfusión pulmonar / Interleukins 1ß, 6 y 10 and tissue damage by pulmonary perfusion and reperfusion

Introducción: La síntesis y la liberación de citocinas se afectan por efecto de la perfusión y de la reperfusión de órganos. Objetivo: En este trabajo, se obtuvo un índice numérico para cada una de las interleucinas detectadas 1b, 6 y 10 en la solución de perfusión y en el tejido pulmonar de bloques pulmonares perfundidos y reperfundidos para correlacionarlo con los hallazgos microscópicos encontrados en dichos bloques. Material y Métodos: Se utilizaron los bloques pulmonares de 21 ratones Balb-C divididos al azar en tres grupos de estudio (n = 7 en cada grupo). Grupos 1: Los bloques fueron perfundidos in situ a través de la arteria pulmonar con solución de Krebs-Henseleit a 4ºC con un flujo de perfusión de 0.2 mL/min únicamente durante el tiempo necesario para exanguinarlos; Grupo 2: Los bloques fueron perfundidos in situ durante 30' a través de la arteria pulmonar con solución de Krebs-Henseleit a 4ºC con un flujo de perfusión de 0.2 mL/min y Grupo 3: Los bloques fueron exanguinados mediante perfusión in situ a través de la arteria pulmonar con solución de Krebs-Henseleit a 4ºC con un flujo de perfusión de 0.2 mL/min, se preservaron durante 24 horas a 4ºC sumergidos en la misma solución y transcurrido el tiempo de isquemia, fueron reperfundidos ex vivo durante 30' con solución de Krebs-Henseleit a 4ºC con un flujo de perfusión, la solución de perfusión se recolectó a través de un catéter conectado en el ventrículo izquierdo. Concluidas las perfusiones y las reperfusiones se disecó el bloque pulmonar, utilizando el resto de los lóbulos para la preparación de homogeneizados. La concentración de las interleucinas en la solución de perfusión y en el homogeneizado tisular, fue determinada mediante la técnica de ELISA. Se realizaron estas concentraciones, calculando un índice para cada interleucina y correlacionando con los hallazgos microscópicos. Resultados: No existen correlaciones importantes entre los índices calculados para cada una de las interleucinas con la mayoria de los hallazgos histológicos, con exepción de que parece existir una correlación negativa entre el índice calculado para la interleucina 10 y la ruptura alveolar por daño mecánico (-0.35), enfisema (-0.38) e inflamación (-0.68, p <0.01)

Superóxido dismutasa, glutatión y malonaldehído en las soluciónes de perfusión y reperfusión pulmonar: estudio experimental murino / Superoxido dismutase, glutathoine and malonaldehide in pulmonary perfusión and reperfusión: Murine experimental study

Introducción: Las especies oxidantes son producto del metabolismo del oxígeno molecular, están reguladas por acción de receptores como la enzima superóxido dismutasa (SOD) y el glutation (GLU). Estos radicales oxidantes pueden generarse durante el periodo de isquemia-reperfusión ocasionando peroxidación lipídica, el malonaldehído (MAL) es un producto de este mecanismo de daño celular. Objetivo: Utilizando un modelo experimental murino de perfusión in situ y reperfusión ex vivo a través del ventrículo derecho, fue evaluada la actividad de SOD, y las concentraciones de GLU y MAL en las soluciones de perfusión y de reperfusión pulmonar recolectadas a través de un catéter colocado en la ventrículo izquierdo, antes y después de preservar el bloque pulmonar durante 24 h a 4ºC en solución de Krebs-Henseleit (KH). Material y métodos: 14 ratones de la cepa Balb-c fueron divididos en dos grupos de estudio. Grupo 1 (n = 7): El bloque pulmonar se perfundió con solución KH únicamente durante el tiempo necesario para exanguinarlo; Grupo 2 (n = 7): Al igual que el Grupo 1, el bloque pulmonar se perfundió con solución KH para exanguinarlo, inmediatamente después se sumergió en solución KH durante 24 h a 4ºC, transcurrido el tiempo de isquemia, el bloque fue reperfundido con solución KH durate 24 H a 4ºC, transcurrido el tiempo de isquemia, el bloque fue reperfundido con solución KH durante 30'. Utilizando estuches comerciales y espectrofotometría, se determinó la actividad de la enzima SOD y las concentraciones de GLU y MAL en las soluciones de perfusión y de reperfusión recolectadas. Resultados: No fueron encontradas diferencias en la actividad de SOD y en las concentraciones de GLU y MAL, obtenidas al exanguinar los bloques en los grupos de estudio 1 y 2. Los tres parámetros evaluados fueron mayores en la solución de perfusión obtenida al exanguinar los bloques antes de preservarlos y disminuyeron significativamente en la solución de reperfusión obtenida post-preservación pulmonar

Nonindependent K+ movement through the pore in IRK1 potassium channels.

We measured unidirectional K+ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K+ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n', of approximately 2.2 and was constant over a voltage range from -50 to -25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n' value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K+ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K+ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current-voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K+ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K+ concentration, and may differ in the number of K+ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.

Interaction of Ba2+ with the pores of the cloned inward rectifier K+ channels Kir2.1 expressed in Xenopus oocytes.

Interactions of Ba2+ with K+ and molecules contributing to inward rectification were studied in the cloned inward rectifier K+ channels, Kir2.1. Extracellular Ba2+ blocked Kir2.1 channels with first-order kinetics in a Vm-dependent manner. At Vm more negative than -120 mV, the Kd-Vm relationship became less steep and the dissociation rate constants were larger, suggesting Ba2+ dissociation into the extracellular space. Both depolarization and increasing [K+]i accelerated the recovery from extracellular Ba2+ blockade. Intracellular K+ appears to relieve Ba2+ blockade by competitively slowing the Ba2+ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100 microM) reduced, whereas 1 mM [Mg2+]i only slightly reduced, the ability of intracellular K+ to repulse Ba2+ from the channel pore. Intracellular Ba2+ also blocked outward IKir2.1 in a voltage-dependent fashion. At Vm >/= +40 mV, where intrinsic inactivation is prominent, intracellular Ba2+ accelerated the inactivation rate of the outward IKir2.1 in a Vm-independent manner, suggesting interaction of Ba2+ with the intrinsic gate of Kir2.1 channels.

Antibody response to Klebsiella pneumoniae 60 kDa protein in familial and sporadic ankylosing spondylitis: role of HLA-B27 and characterization as a GroEL-like protein.

OBJECTIVE: To study the antibody response of HLA-B27+ patients with ankylosing spondylitis (AS) and their first degree relatives to the 60 kDa protein of Klebsiella pneumoniae and to characterize this protein. METHODS: Sera from 84 individuals were analyzed by ELISA to determine the titer of antibodies against the 60 kDa protein of K. pneumoniae. Subjects were divided into 3 categories: Group 1: 44 HLA-B27+ AS related individuals (35 patients, 9 healthy controls); Group 2: 28 healthy B27- AS related individuals; and Group 3: 12 healthy B27- non-AS related subjects. The 60 kDa protein of K. pneumoniae was induced at 45 degrees C and purified by electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was characterized as a GroEL-like heat shock protein (HSP). The recognition of GroEL-like protein was confirmed by immunoblot of 2 dimension electrophoresis. The response to GroEL-like protein from other bacteria and the response to lipopolysaccharide (LPS) was also analyzed by immunoblot. RESULTS: HLA-B27+ individuals (Group 1), independent of their disease status, showed a significant higher response to the 60 kDa protein of K. pneumoniae than HLA-B27- subjects from Groups 2 and 3 (p < 0.0001). This protein was characterized as a HSP of the GroEL family and designated HSP60Kp. The GroEL of other enterobacteria as well as that of Mycobacterium leprae were recognized by HLA-B27+ individuals by immunoblot, whereas HLA-B27- individuals did not. LPS was not recognized by HLA-B27 positive or negative subjects. CONCLUSION: These findings suggest a relationship between HLA-B27 and the response to a GroEL-like protein that could have implications in AS.

Inflammatory cells and ferruginous bodies in bronchoalveolar lavage in urban dogs.

OBJECTIVE: To investigate whether high levels of atmospheric pollution modify the inflammatory cell count of the canine lung and to detect the presence of ferruginous bodies (FB) in the respiratory system. STUDY DESIGN: Bronchoalveolar lavage (BAL) was performed on dogs from four different areas of Mexico City and a rural area. With the BAL fluid recovered, total and differential cell counts were made, and ferruginous bodies were isolated by sodium hypochlorite digestion. They were counted by light microscopy and evaluated as a marker of exposure to particulate pollutants. RESULTS: There was no significant difference in the total cell count or in the macrophage number between the five groups. The neutrophil count was statistically higher in dogs from the southwest area as compared to the northeast and rural areas (P < .05). The lymphocyte count was significantly greater in the southwest, also. The northeast part of the city showed the highest number of FB in BAL fluids. CONCLUSION: The most polluted areas, in terms of particulate matter, were the northeast and the northwest; those are the locations of heavy industry. In contrast, in the southwest, where more inflammation was seen, the highest levels of ozone were registered during the year.

Comparison of voltage-activated Cl- channels in rat parotid acinar cells with ClC-2 in a mammalian expression system.

Rat parotid acinar cells express Cl- currents that are activated in a time-dependent manner by hyperpolarized potentials. ClC-2, a member of the ClC gene family, codes for a voltage-gated, inward rectifying anion channel when expressed in Xenopus oocytes. In the present study, we found that cDNA derived from individual parotid acinar cells contained sequence identical to that reported for ClC-2 in rat brain and heart. A polyclonal antibody generated against the N-terminal cytoplasmic domain of ClC-2 recognized an approximately 100 kD protein on western blots of both brain and parotid gland. ClC-2 expressed in oocytes has different kinetics from the currents found in parotid acinar cells. Since the ClC-2 channel was cloned from and its transcripts are expressed in mammalian tissue, we compared the channel properties of acinar cells to a mammalian expression system. We expressed ClC-2 channels in human embryonic kidney cells, HEK 293, using recombinant ClC-2 DNA and ClC-2 DNA fused with DNA coding for jellyfish green fluorescent protein (GFP). Confocal microscopy revealed that the expressed ClC-2-GFP chimera protein localized to the plasma membrane. Whole cell Cl- currents from HEK 293 cells expressing ClC-2-GFP were similar, if not identical, to the Cl- currents recorded from cells transfected with ClC-2 cDNA (no GFP). The voltage-dependence and kinetics of ClC-2 channels expressed in HEK 293 cells were quite similar to those in acinar cells. Channels in parotid acinar and HEK 293 cells activated at more positive membrane potentials and with a faster time course than the channels expressed in Xenopus oocytes. In summary, we found that ClC-2 message and protein are expressed in salivary cells and that the properties of voltage-activated, inward rectifying Cl- channels in acinar cells are similar to those generated by the ClC-2-GFP construct expressed in HEK 293 cells. The properties of the ClC-2 anion channel seem to be dependent on the type of cell background in which it is expressed.

Differences in regulation of Ca(2+)-activated Cl- channels in colonic and parotid secretory cells.

We investigated the regulation of Ca(2+)-activated Cl- channels in cells from the human colonic cell line T84 and acinar cells from rat parotid glands. The participation of multifunctional Ca(2+)- and calmodulin-dependent protein kinase (CaM kinase) II in the activation of these channels was studied using selective inhibitors of calmodulin and CaM kinase II. Ca(2+)-dependent Cl- currents were recorded using the whole cell patch-clamp technique. Direct inhibition of CaM kinase II by 40 microM peptide 281-302 or by 10 microM KN-62, another CaM kinase inhibitor, did not block the Cl- current in parotid acinar cells, whereas in T84 cells KN-62 markedly inhibited the Ca(2+)-dependent Cl- current. We also used the calmodulin-binding domain peptide 290-309 (0.5 microM), which competitively inhibits the activation of CaM kinase II. This peptide reduced the Cl- current in T84 cells by approximately 70% but was without effect on the channels in parotid acinar cells. We conclude that the Ca(2+)-dependent Cl- channels in T84 cells are activated by CaM kinase II but that the channels in parotid acinar cells must be regulated by a fundamentally different Ca(2+)-dependent mechanism that does not utilize CaM kinase II or any calmodulin-dependent process.

Activation of calcium-dependent chloride channels in rat parotid acinar cells.

The Ca2+ and voltage dependence of Ca(2+)-activated Cl- currents in rat parotid acinar cells was examined with the whole-cell patch clamp technique. Acinar cells were dialyzed with buffered free Ca2+ concentrations ([Ca2+]i) from < 1 nM to 5 microM. Increasing [Ca2+]i induced an increase in Cl- current at all membrane potentials. In cells dialyzed with [Ca2+]i > 25 nM, depolarizing test pulses activated a Cl- current that was composed of an instantaneous and a slow monoexponential component. The steady-state current-voltage relationship showed outward rectification at low [Ca2+]i but became more linear as the [Ca2+]i increased because of a shift in Cl- channel activation toward more negative voltages. The Ca2+ dependence of steady-state channel activation at various membrane voltages was fit by the Hill equation. The apparent Kd and Hill coefficient obtained from this analysis were both functions of membrane potential. The Kd decreased from 417 to 63 nM between -106 and +94 mV, whereas the Hill coefficient was always > 1 and increased to values as large as 2.5 at large positive potentials. We found that a relatively simple mechanistic model can account for the channel steady-state and kinetic behavior. In this model, channel activation involves two identical, independent, sequential Ca2+ binding steps before a final Ca(2+)-independent transition to the conducting conformation. Channel activation proceeds sequentially through three closed states before reaching the open state. The Ca2+ binding steps of this model have a voltage dependence similar to that of the Kd from the Hill analysis. The simplest interpretation of our findings is that these channels are directly activated by Ca2+ ions that bind to sites approximately 13% into the membrane electric field from the cytoplasmic surface.

Antibody response to nitrogenase-positive and -negative Klebsiella pneumoniae strains in juvenile-onset ankylosing spondylitis patients and their first degree relatives: lack of differential recognition of the bacterial nitrogenase.

In the search for the pathogenic consequences of the molecular mimicry between the Klebsiella pneumoniae nitrogenase and the HLA-B27 antigen, sera from individuals belonging to 16 kindreds with juvenile-onset ankylosing spondylitis cases, were analyzed for antibodies against nitrogenase-positive and -negative K. pneumoniae whole bacterial extracts. An initial screening for nitrogenase producing K. pneumoniae strains was performed in 31 clinical isolates. The best nitrogenase producing strain was selected as well as a non producing one for immunoblot analysis using sera from 82 subjects, 55 HLA-B27 positive, of which 26 had some clinical manifestations. Even though electrophoretic patterns were different in both strains, there was no distinctive differential recognition of the 30-40 kDa proteins where the nitrogenase subcomponent which shares the sequence QTDRED with the HLA-B27 molecule is located. On the other hand, strong recognition of a protein of 60 kDa (p60Kp) was detected in 75% of HLA-B27 positive tested subjects independently of their clinical status. Studies on the nature of this protein and its participation in the pathogenesis of ankylosing spondylitis are now in progress.

Three distinct chloride channels control anion movements in rat parotid acinar cells.

1. We used the whole-cell configuration of the patch clamp technique to examine the different macroscopic Cl- currents present in single rat parotid acinar cells. 2. Cell swelling produced by negative osmotic pressure (hypotonic bath solutions) induced a large outwardly rectifying Cl- current with little or no time and voltage dependence. In contrast, an increase in intracellular [Ca2+] induced by ionomycin activated Cl- currents with very different properties. Ca(2+)-activated Cl- currents showed outward rectification, relatively slow activation kinetics and marked voltage dependence. These results are consistent with the existence of two different outwardly rectifying Cl- channels in rat parotid cells. 3. In conditions designed to eliminate the activation of these two Cl- currents, a third type of current was observed. This third current was activated in a time-dependent manner by hyperpolarized potentials and was about equally permeant to Cl-, I- and Br-. 4. The properties of the hyperpolarization-activated current were similar to those of the cloned ClC-2 channel. Polymerase chain reaction-based methods and ribonuclease protection analyses indicated the presence in parotid gland of mRNA homologous to ClC-2. 5. Individual parotid acinar cells expressed all three types of Cl- channels. Each type of channel may contribute to Cl- efflux in distinct stages of the secretion process depending on the intracellular [Ca2+], cell volume and membrane potential.

Thoracoabdominal wall repair with glutaraldehyde-preserved bovine pericardium.

Glutaraldehyde-preserved bovine pericardium (GPBP) is evaluated as a bioprosthesis for the reconstruction of surgical defects in the thoracoabdominal wall. The mechanical properties of bovine pericardium preserved at different concentrations of glutaraldehyde were studied. Samples preserved in 0.5% glutaraldehyde showed a significantly higher tensile strength (11.7 +/- 0.8 N/mm2) than samples preserved in 2.5, 5, or 10% (similar to pericardium preserved in normal saline). The percentage of elongation was significantly lower than samples preserved in 1, 2.5, and 5% glutaraldehyde. GPBP at 0.5% was used to repair experimentally induced defects of the abdominal wall (n = 9), chest wall (n = 6), diaphragm (n = 6), and sternum (n = 7). All animals presented adequate tolerance to the material used and no case of infection or rejection of the material was seen in any of the animals. Finally, 0.5% GPBP was used clinically in a series of 40 patients: postincisional abdominal hernia (n = 30), inguinal hernia (n = 8), diaphragmatic hernia (n = 1), and congenital pelvic defect with prolapse of abdominal organs (n = 1). Surgical use showed that GPBP was a very manageable material and long-term results were good in 37 patients with a mean follow up of 18 months (range 5-35 months). Six patients presented seroma formation (all abdominal hernia patients), three of which eventually developed infection and had the GPBP patch removed at 3, 5, and 7 months postoperatively. The rest of the patients presented good scar formation with adequate resistance at the area of implantation. GPBP is a biological material with sufficient resistance to be used surgically in the repair of thoracoabdominal defects. Ideal concentration of glutaraldehyde to be used in the preparation-preservation of the material is 0.5% since higher concentration negatively affect its tensile rupture strength and elongation.