Gap junctions in the cardiovascular and immune systems

Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system.

Gap junctions in cells of the immune system: structure, regulation and possible functional roles

Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90%) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium.

A moderate decrease in temperature inhibits the calcium signaling mechanism(s) of the regulatory volume decrease in chick embryo cardiomyocytes

Chick cardiomyocytes, when submitted to hyposmotic swelling, exhibit a partial regulatory volume decrease (RVD). A Ca2+ influx by stretch-activated channels signals a taurine efflux and the RVD at 37°C. We evaluated the cell's performance at room temperature. Cardiomyocytes isolated and cultured from 11-day-old chick embryos were submitted to a hyposmotic solution (180 mOsm/kg H2O) at 37°C and at room temperature (26°C). Under these conditions we measured the changes in cell volume as well as the intracellular free Ca2+ (using fura-2). During hyposmotic swelling, cells at 37°C displayed a peak relative volume of 1.61 ± 0.03 and recovery to 1.22 ± 0.04 (N = 14), while cells at 26°C presented a peak swell relative volume of 1.74 ± 0.06 and did not recover (1.59 ± 0.09, N = 9). Transient increases in intracellular Ca2+, which are characteristic of the normal RVD, were observed at both temperatures (29.1 ± 4.5% (N = 8) and 115.2 ± 42.8% (N = 5) increase at 37° and 26°C (P<0.05), respectively). A delay in the Ca2+ transient increase was also observed when the cells were at 26°C (109 ± 34 s compared to 38 ± 9 s at 37°C, P<0.05). At room temperature the RVD does not occur because the calcium transient increase, which is an early event in the signaling of the RVD, is delayed. Also, free calcium is not cleared as in the 37°C RVD. In the normal RVD the free calcium returns to baseline levels. The very high and persistent free calcium levels seen at room temperature can lead to unregulated enzyme activities and may promote irreversible injury and cell death.

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 µmol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 µmol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children.

Uniform decrease of alpha-global field power induced by intermittent photic stimulation of healthy subjects

Nineteen-channel EEGs were recorded from the scalp surface of 30 healthy subjects (16 males and 14 females, mean age: 34 years, SD: 11.7 years) at rest and under trains of intermittent photic stimulation (IPS) at rates of 5, 10 and 20 Hz. Digitalized data were submitted to spectral analysis with fast fourier transformation providing the basis for the computation of global field power (GFP). For quantification, GFP values in the frequency ranges of 5, 10 and 20 Hz at rest were divided by the corresponding data obtained under IPS. All subjects showed a photic driving effect at each rate of stimulation. GFP data were normally distributed, whereas ratios from photic driving effect data showed no uniform behavior due to high interindividual variability. Suppression of alpha-power after IPS with 10 Hz was observed in about 70% of the volunteers. In contrast, ratios of alpha-power were unequivocal in all subjects: IPS at 20 Hz always led to a suppression of alpha-power. Dividing alpha-GFP with 20-Hz IPS by alpha-GFP at rest (R = alpha-GFP IPS/alpha-GFPrest) thus resulted in ratios lower than 1. We conclude that ratios from GFP data with 20-Hz IPS may provide a suitable paradigm for further investigations.

Immune response induced in mice oral immunization with cowpea severe mosaic virus

There is increasing interest in the immune response induced by plant viruses since these could be used as antigen-expressing systems in vaccination procedures. Cowpea severe mosaic virus (CPSMV), as a purified preparation (300 g of leaves, 2 weeks post-inoculation), or crude extract from cowpea (Vigna unguiculata) leaves infected with CPSMV both administered by gavage to Swiss mice induced a humoral immune response. Groups of 10 Swiss mice (2-month-old females) were immunized orally with 10 daily doses of either 50 µg viral capsid protein (boosters of 50 µg at days 21 and 35 after immunization) or 0.6 mg protein of the crude extract (boosters of 0.6 mg at days 21 and 35 after immunization). Anti-CPSMV antibodies were quantified by ELISA in pooled sera diluted at least 1:400 at days 7, 14, 21, 28, 35 and 42 after the 10th dose. IgG and IgA against CPSMV were produced systemically, but IgE was not detected. No synthesis of specific antibodies against the proteins of leaf extracts from V. unguiculata, infected or not with CPSMV, was detected. The use of CPSMV, a plant-infecting virus that apparently does not induce a pathogenic response in animals, induced a humoral and persistent (at least 6 months) immune response through the administration of low antigen doses by gavage. These results raise the possibility of using CPSMV either as a vector for the production of vaccines against animal pathogens or in quick and easy methods to produce specific antisera for viral diagnosis.

Immune response to respiratory syncytial virus in young Brazilian children

We have evaluated the cellular and humoral immune response to primary respiratory syncytial virus (RSV) infection in young infants. Serum specimens from 65 patients <=12 months of age (39 males and 26 females, 28 cases <3 months and 37 cases > or = 3 months; median 3 ± 3.9 months) were tested for anti-RSV IgG and IgG subclass antibodies by EIA. Flow cytometry was used to characterize cell surface markers expressed on peripheral blood mononuclear cells (PBMC) from 29 RSV-infected children. There was a low rate of seroconversion in children <3 months of age, whose acute-phase PBMC were mostly T lymphocytes (63.0 ± 9.0%). In contrast, a higher rate of seroconversion was observed in children >3 months of age, with predominance of B lymphocytes (71.0 ± 17.7%). Stimulation of PBMC with RSV (2 x 10(5) TCID50) for 48 h did not induce a detectable increase in intracellular cytokines and only a few showed a detectable increase in RSV-specific secreted cytokines. These data suggest that age is an important factor affecting the infants' ability to develop an immune response to RSV.

Indirect effects of oral tolerance to ovalbumin interfere with the immune responses triggered by Schistosoma mansoni eggs

The objective of the present study was to investigate whether the injection of a tolerated protein (indirect effects) affects the formation of granulomas around Schistosoma mansoni eggs trapped in the lungs after intravenous (iv) injection into normal (noninfected) C57BL/6 mice (6 animals per group). To induce oral tolerance to chicken egg ovalbumin a 1/5 dilution of egg white in water was offered ad libitum in a drinking bottle for 3 days. Control mice received water. After 7 days, control and experimental animals were injected iv with 2,000 S. mansoni eggs through a tail vein. In some mice of both groups the iv injection of eggs was immediately followed by intraperitoneal (ip) immunization with 10 µg of dinitrophenylated conjugates of ovalbumin (DNP-Ova) emulsified in complete Freund's adjuvant (CFA) or only CFA; 18 days later, mice were bled and killed by ether inhalation. The lungs were fixed in formalin and embedded in paraffin. Serial sections of 5 µm were stained with Giemsa, Gomori's silver reticulin and Sirius red (pH 10.2). Granuloma diameters were measured in histological sections previously stained with Gomori's reticulin. Anti-DNP and anti-soluble egg antigen (SEA) antibodies were analyzed by ELISA. In mice orally tolerant to ovalbumin the concomitant ip injection of DNP-Ova resulted in significantly lower anti-SEA antibodies (ELISA*: 1395 ± 352 in non-tolerant and 462 ± 146 in tolerant mice) and affected granuloma formation around eggs, significantly decreasing granuloma size (area: 22,260 ± 2478 to 12,993 ± 3242 µm²). Active mechanisms triggered by injection of tolerated antigen (ovalbumin) reduce granuloma formation.

The decrease in uroporphyrinogen decarboxylase activity induced by ethanol predisposes rats to the development of porphyria and accelerates xenobiotic-triggered porphyria, regardless of hepatic damage

We evaluated the porphyrinogenic ability of ethanol (20% in drinking water) per se, its effect on the development of sporadic porphyria cutanea tarda induced by hexachlorobenzene in female Wistar rats (170-190 g, N = 8/group), and the relationship with hepatic damage. Twenty-five percent of the animals receiving ethanol increased up to 14-, 25-, and 4.5-fold the urinary excretion of delta-aminolevulinate, porphobilinogen, and porphyrins, respectively. Ethanol exacerbated the precursor excretions elicited by hexachlorobenzene. Hepatic porphyrin levels increased by hexachlorobenzene treatment, while this parameter only increased (up to 90-fold) in some of the animals that received ethanol alone. Ethanol reduced the activities of uroporphyrinogen decarboxylase, delta-aminolevulinate dehydrase and ferrochelatase. In the ethanol group, many of the animals showed a 30% decrease in uroporphyrinogen activity; in the ethanol + hexachlorobenzene group, this decrease occurred before the one caused by hexachlorobenzene alone. Ethanol exacerbated the effects of hexachlorobenzene, among others, on the rate-limiting enzyme delta-aminolevulinate synthetase. The plasma activities of enzymes that are markers of hepatic damage were similar in all drug-treated groups. These results indicate that 1) ethanol exacerbates the biochemical manifestation of sporadic hexachlorobenzene-induced porphyria cutanea tarda; 2) ethanol per se affects several enzymatic and excretion parameters of the heme metabolic pathway; 3) since not all the animals were affected to the same extent, ethanol seems to be a porphyrinogenic agent only when there is a predisposition, and 4) hepatic damage showed no correlation with the development of porphyria cutanea tarda.

The conservative physiology of the immune system

Current immunological opinion disdains the necessity to define global interconnections between lymphocytes and regards natural autoantibodies and autoreactive T cells as intrinsically pathogenic. Immunological theories address the recognition of foreignness by independent clones of lymphocytes, not the relations among lymphocytes or between lymphocytes and the organism. However, although extremely variable in cellular/molecular composition, the immune system preserves as invariant a set of essential relations among its components and constantly enacts contacts with the organism of which it is a component. These invariant relations are reflected, for example, in the life-long stability of profiles of reactivity of immunoglobulins formed by normal organisms (natural antibodies). Oral contacts with dietary proteins and the intestinal microbiota also result in steady states that lack the progressive quality of secondary-type reactivity. Autoreactivity (natural autoantibody and autoreactive T cell formation) is also stable and lacks the progressive quality of clonal expansion. Specific immune responses, currently regarded as the fundament of the operation of the immune system, may actually result from transient interruptions in this stable connectivity among lymphocytes. More permanent deficits in interconnectivity result in oligoclonal expansions of T lymphocytes, as seen in Omenn's syndrome and in the experimental transplantation of a suboptimal diversity of syngeneic T cells to immunodeficient hosts, which also have pathogenic consequences. Contrary to theories that forbid autoreactivity as potentially pathogenic, the physiology of the immune system is conservative and autoreactive. Pathology derives from failures of these conservative mechanisms.

Effect of surgical treatment on the cellular immune response of gastric cancer patients

Patients with gastric cancer have a variety of immunological abnormalities. In the present study the lymphocytes and their subsets were determined in the peripheral blood of patients with gastric cancer (N = 41) both before and after surgical treatment. The percent of helper/inducer CD4 T cells (43.6 ± 8.9) was not different after tumor resection (43.6 ± 8.2). The percent of the cytotoxic CD8+ T cell population decreased significantly, whether patients were treated surgically (27.2 ± 5.8%, N = 20) or not (27.3 ± 7.3%, N = 20) compared to individuals with inflammatory disease (30.9 ± 7.5%) or to healthy individuals (33.2 ± 7.6%). The CD4/CD8 ratio consequently increased in the group of cancer patients. The peripheral blood lymphocytes of gastric cancer patients showed reduced responsiveness to mitogens. The defective blastogenic response of the lymphocytes was not associated with the production of transforming growth factor beta (TGF-ß) since the patients with cancer had reduced production of TGF-ß1 (269 ± 239 pg/ml, N = 20) in comparison to the normal individuals (884 ± 175 pg/ml, N = 20). These results indicate that the immune response of gastric cancer patients was not significantly modified by surgical treatment when evaluated four weeks after surgery and that the immunosuppression observed was not due to an increase in TGF-ß1 production by peripheral leukocytes.

The role of complement in the modulation by fluid-phase IgG of the production of reactive oxygen species by polymorphonuclear leukocytes stimulated with IgG immune complexes

The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMN) can be induced by immune complexes and is an important component of phagocytosis in the killing of microorganisms, but can also be involved in inflammatory reactions when immune complexes are deposited in tissues. We have observed that fluid-phase IgG can inhibit the generation of ROS by rabbit PMN stimulated with precipitated immune complexes of IgG (ICIgG) in a dose-dependent manner, acting as a modulatory factor in the range of physiological IgG concentrations. This inhibitory effect is compatible with the known affinity (Kd) of monomeric IgG for the receptors involved (FcRII and FcRIII). The presence of complement components in the immune complexes results in a higher stimulation of ROS production. In this case, however, there is no inhibition by fluid-phase IgG. The effect of complement is strongly dependent on the presence of divalent cations (Ca2+ or Mg2+) in the medium, whereas the stimulation of ICIgG (without complement) does not depend on these cations. We have obtained some evidence indicating that iC3b should be the component involved in the effect of complement through interaction with the CR3 receptor. The absence of the inhibitory effect of fluid-phase IgG in ROS production when complement is present in the immune complex shows that complement may be important in vivo not only in the production of chemotactic factors for PMN, but also in the next phase of the process, i.e., the generation of ROS.

Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting ß-cell regeneration

Type 1 diabetes mellitus results from a cell-mediated autoimmune attack against pancreatic ß-cells. Traditional treatments involve numerous daily insulin dosages/injections and rigorous glucose control. Many efforts toward the identification of ß-cell precursors have been made not only with the aim of understanding the physiology of islet regeneration, but also as an alternative way to produce ß-cells to be used in protocols of islet transplantation. In this review, we summarize the most recent studies related to precursor cells implicated in the regeneration process. These include embryonic stem cells, pancreas-derived multipotent precursors, pancreatic ductal cells, hematopoietic stem cells, mesenchymal stem cells, hepatic oval cells, and mature ß-cells. There is controversial evidence of the potential of these cell sources to regenerate ß-cell mass in diabetic patients. However, clinical trials using embryonic stem cells, umbilical cord blood or adult bone marrow stem cells are under way. The results of various immunosuppressive regimens aiming at blocking autoimmunity against pancreatic ß-cells and promoting ß-cell preservation are also analyzed. Most of these regimens provide transient and partial effect on insulin requirements, but new regimens are beginning to be tested. Our own clinical trial combines a high dose immunosuppression with mobilized peripheral blood hematopoietic stem cell transplantation in early-onset type 1 diabetes mellitus.