Neo-adjuvant chemo/immunotherapy in the treatment of stage III (N2) non-small cell lung cancer: a phase I/II pilot study.

In a previous randomized study, we showed that adjuvant immunotherapy with tumor-infiltrating lymphocytes and recombinant interleukin-2 (rIL-2) significantly improved survival in resected N2-non small cell lung cancer (NSCLC) patients. The present study assesses feasibility, safety and potential efficacy of combined neo-adjuvant chemotherapy and immunotherapy with peripheral blood mononuclear cells (PBMC) and rIL-2 in resectable N2-NSCLC patients. Eighty-two consecutive N2-NSCLC patients underwent neo-adjuvant chemotherapy with cisplatin and gemcitabine. Out of the 82 patients, 23 were also subjected to leukapheresis prior to neo-adjuvant chemotherapy while the remaining 59 did not. Collected PBMC were analyzed for viability and phenotype and then stored frozen in liquid nitrogen. Thawed PBMC were infused intravenously, 5 days before surgery. After the infusion, rIL-2 was administered subcutaneously until surgery. Only patients with a partial or complete response to neoadjuvant chemotherapy underwent surgery: 13 patients in the experimental immunotherapy group (A) and 32 in the reference group (B). The two groups were homogeneous for all major prognostic factors. Median leukapheresis yield was 10 billion PBMC, (range 3-24 billions). Two to six billion PBMC were infused. The phenotypic analysis showed that similar proportions of CD4 and CD8 cells were present in leukapheresis products, and thawed PBMC, as well as in T lymphocytes isolated from the removed tumours. No severe adverse effects were observed following immunotherapy. No significant differences in overall survival (OS) and event-free survival (EFS) were seen between the two groups. However, the 5-year OS in group A was almost twice as much compared to group B (59 percent vs 32 percent). After adjustment for major prognostic factors, a statistically significant 66 percent reduction in the hazard of death was seen in patients receiving immunotherapy. The OS benefit was more evident in patients with adenocarcinoma than in those with squamous cell carcinoma. This study supports the favorable toxicity profile and potential efficacy of combining neo-adjuvant chemotherapy and immunotherapy with PBMC and rIL-2 in the treatment of N2-NSCLC patients.

CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta.

The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells.

Identification of four subsets of human CD3-CD16+ natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition.

In previous studies we identified a surface molecule (termed GL183) capable of mediating cell activation and selectively expressed by a subset of human CD3-CD16+ natural killer (NK) cells. In this study we analyzed whether other subset-specific functional molecules were expressed in GL183- NK cells. To this end, mice were immunized with the PE29 (CD3-CD16+GL183-) NK clone. Monoclonal antibodies (mAbs) were selected by screening the hybridoma supernatants for their ability to trigger the cytolytic activity of clone PE29 against the human myelomonocytic leukemia U937. The EB6 mAb (IgG1) triggered the PE29 clone, but not a GL183+ clone used as a control. EB6+ cells ranged between 1 and 13% of peripheral blood lymphocytes and were largely included in the CD3-CD16+CD56+ cell populations (only less than 2% of EB6+ cells were CD3+). Analysis of resting or activated CD3-CD16+ populations, or clones for the expression of EB6 or GL183 mAbs, allowed us to identify four distinct, phenotypically stable, NK subsets (EB6+GL183-; EB6+GL183+; EB6-GL183+; EB6-GL183-). Similar to GL183 mAb, the EB6 mAb selectively triggered the NK subset expressing the corresponding surface antigen to lyse human tumor cell lines including U937, IGROV-I, M14, and A549. In addition, EB6 mAb sharply inhibited the cytolytic activity of EB6+ clones against P815, M12, and P3U1 murine target cells. In EB6+GL183+ ("double-positive") clones both EB6 and GL183 mAb inhibited the redirected killing of P815 cells induced by anti-CD16, anti-CD2 mAbs and phytohemagglutinin (PHA). Similar to GL183 molecules, molecules precipitated by EB6 mAb were represented by either single 58-kD chain or double chains of 55 and 58 kD (with no detectable differences in EB6+GL183- or EB6+GL183+ clones). In sequential immunoprecipitation experiments using the double-positive clones CEG52 and CA25.50, preclearing of cell lysates with EB6 or GL183 mAb removed only EB6 or GL183 molecules, respectively, thus indicating that the two antigenic determinants are carried by two distinct molecules. Peptide map analysis indicated that EB6 (or GL183) molecules precipitated from double-positive clones were identical to the corresponding molecules isolated from single-positive ones. On the other hand, comparison of the EB6 and GL183 maps revealed peptides that were unique to each molecule, although most of the major peptides migrated to identical positions. We further investigated whether correlation existed between the phenotypic assignment of NK clones and their ability to mediate specific lysis of normal allogeneic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel surface antigen expressed by a subset of human CD3- CD16+ natural killer cells. Role in cell activation and regulation of cytolytic function.

The GL183 mAb was obtained by immunizing BALB/c mice with the E57 clone (CD7+CD2+CD3-CD16+CD56+) derived from human peripheral blood NK cells. In human peripheral blood, GL183-reactive cells ranged between 2 and 12% (mean 6.5%) in 10 different donors. Double fluorescence and FACS analysis showed that GL183+ cells were consistently included in the CD56+ or CD16+ cell populations. Moreover, since only a fraction of CD56+ or CD16+ cells (approximately 40%) coexpressed GL183 surface antigen, reactivity with GL183 mAb appears to define two subsets within the CD3- lymphocyte population expressing NK cell markers. Although, the majority of GL183+ cells were CD3-, approximately 1% expressed CD3 surface antigens. As shown by clonal analysis, these infrequent CD3+GL183+ cells coexpressed CD56 and CD16 antigens. Cloning of CD3-GL183+ or CD3-GL183- cell populations under limiting dilution conditions yielded clonal progenies that maintained their original surface phenotype. Therefore, expression or lack of expression of GL183 surface antigens represents a stable phenotypic property of a subset of human CD3- NK cells. Immunoprecipitation experiments and two-dimensional PAGE analysis indicated that GL183-reactive molecules were represented in different clones either by a single 58-kD chain or, more frequently, by two chains of approximately 55 and approximately 58 kD, respectively. Analysis of GL183+ or GL183- NK clones for their ability to lyse human (IGROV I) or murine (P815) tumor target cells indicated that GL183- clones were, on average, fivefold more efficient in inducing target cell lysis. GL183+ and GL183- clones produced comparable levels of TNF-alpha in response to PHA plus PMA or anti-CD16 mAb plus PMA. Importantly, production of TNF-alpha was also induced by stimulation of GL183+ clones with GL183 mAb plus PMA. These data indicated that GL183 antigen could mediate cell triggering. This concept was confirmed by the analysis of Ca2+ mobilization, as GL183 mAb induced (in GL183+ clones) increments of [Ca2+]i comparable with those induced by PHA. Moreover, GL183 mAb, or its F(ab')2 fragments, strongly enhanced the cytolytic activity of GL183+ clones against a panel of human tumor target cells, including U937, Raji, IGROV I, M14, and A549. In contrast, GL183 mAb, but not the F(ab')2 fragments, sharply inhibited the cytolytic activity of the same clones against P815, M12, and P3U1 murine target cells. In this case, the effect of GL183 mAb (inhibition) was opposite that of PHA or of stimulatory anti-CD2 or anti-CD16 mAbs, which consistently enhanced the target cell lysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence of a natural killer (NK) cell repertoire for (allo) antigen recognition: definition of five distinct NK-determined allospecificities in humans.

Previous studies indicated that CD3-CD16+ natural killer (NK) cells are capable of specific alloantigen recognition. Thus, alloreactive NK clones lysed normal allogeneic target cells (phytohemagglutinin [PHA] blasts) bearing the stimulating alloantigen but did not lyse autologous cells or the majority of unrelated allogeneic cells. In this study we investigated whether NK cells isolated from single individuals could exhibit different allospecificities. To this end, we derived large numbers of CD3-CD16+ clones (in the presence of PHA) from fresh CD3- peripheral blood lymphocytes. Cloning efficiencies ranged between 5 and 10%. The resulting CD3-CD16+ clones were tested for their reactivity against a panel of allogeneic PHA blasts (derived from six donors). In a given individual (A), four distinct groups of clones could be identified according to their pattern of reactivity (over 400 clones have been analyzed). Clones that could be assigned to one or another group of specificity represented 36% of all clones derived from this donor. The remaining clones did not display cytolytic activity against any of the allogeneic target cells used in the panel. None of the clones lysed autologous (A) PHA blasts, yet, these cells were lysed by the representative clones G10 and H12 specific for donor A. Clones displaying a cytolytic pattern of reactivity identical to that defined for donor A were present in other individuals studied, however not all groups of allospecific clones were necessarily represented in different individuals. Allospecific clones belonging to the various groups were homogeneous in the expression of EB6/GL183-triggering surface molecules, and could thus be assigned to one or another of the previously defined subsets of NK cells. Genetic analysis of the new NK-defined alloantigens was performed in representative families. The corresponding characters were found to segregate independently and, at least for three of them, an autosomic recessive type of inheritance could be demonstrated. Moreover, the comparative analysis of the segregation of the major histocompatibility complex haplotypes and the recessive or dominant alleles of the genes governing the five specificities analyzed indicated that there is no independent sampling between the two genetic traits, thus suggesting that the genes regulating the NK-defined specificities are carried by chromosome 6. Finally, some donors expressed more than one specificity, thus providing evidence for an NK-defined complex haplotype.

Incidence of bacteremias and invasive mycoses in children undergoing allogeneic hematopoietic stem cell transplantation: a single center experience.

We performed a retrospective single center study to define the epidemiology of bacteremias or invasive mycoses in pediatric allogeneic hematopoietic SCT (HSCT) from matched related donors (MRD) or alternative donors (AD). During 119 213 days of follow-up, 156 infections were observed: 130 bacteremias (27 in MRD-HSCT and 103 in AD-HSCT recipients) and 26 invasive mycoses (8 in MRD-HSCT and 18 in AD-HSCT recipients). Overall, the risk of bacteremia was fivefold that of invasive mycosis (P<0.001). AD-HSCT recipients had a higher percentage of infections (89 vs 27%; P<0.001), a higher rate/100 days of immunosuppression (infection rate (IR): 0.21 vs 0.06; P<0.001) and a higher proportion of repeated infections (44 vs 9%; P=0.001). In AD-HSCT, the relative risk of bacteremia was 2.87 in the pre-engraftment period, 5.84 in the early post-engraftment period and 6.46 in the late post-engraftment period (P<0.001) compared to MRD-HSCT. Only after 1 year did the epidemiology become similar. The epidemiology of invasive mycoses did not differ significantly between the two types of transplant.

Hypertension-associated point mutations in the adducin alpha and beta subunits affect actin cytoskeleton and ion transport.

The adducin heterodimer is a protein affecting the assembly of the actin-based cytoskeleton. Point mutations in rat adducin alpha (F316Y) and beta (Q529R) subunits are involved in a form of rat primary hypertension (MHS) associated with faster kidney tubular ion transport. A role for adducin in human primary hypertension has also been suggested. By studying the interaction of actin with purified normal and mutated adducin in a cell-free system and the actin assembly in rat kidney epithelial cells (NRK-52E) transfected with mutated rat adducin cDNA, we show that the adducin isoforms differentially modulate: (a) actin assembly both in a cell-free system and within transfected cells; (b) topography of alpha V integrin together with focal contact proteins; and (c) Na-K pump activity at V(max) (faster with the mutated isoforms, 1281 +/- 90 vs 841 +/- 30 nmol K/h.mg pt., P < 0.0001). This co-modulation suggests a role for adducin in the constitutive capacity of the epithelia both to transport ions and to expose adhesion molecules. These findings may also lead to the understanding of the relation between adducin polymorphism and blood pressure and to the development of new approaches to the study of hypertension-associated organ damage.

Zoledronic acid boosts γδ T-cell activity in children receiving αß+ T and CD19+ cell-depleted grafts from an HLA-haplo-identical donor.

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αß+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αß T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

Whole-genome sequencing as standard practice for the analysis of clonality in outbreaks of meticillin-resistant Staphylococcus aureus in a paediatric setting.

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-associated infections. This study investigated the potential use of whole-genome sequencing (WGS) for surveillance purposes by re-examining MRSA strains related to past outbreaks among hospitalized paediatric patients. WGS data ameliorated the genotypic profile previously obtained with Sanger sequencing and pulsed-field gel electrophoresis typing, and discriminated between strains that were related and unrelated to the outbreaks. This allowed strain clonality to be defined with a higher level of resolution than achieved previously. This study demonstrates the potential of WGS to trace hospital outbreaks, which may lead to WGS becoming standard practice in outbreak investigations.

Ouabain antagonists as antihypertensive agents.

The evidence that high levels of endogenous ouabain (EO), a closely related isomer of ouabain, are implicated in human hypertension and cardiac hypertrophy and failure stimulated the pharmacological research for developing novel anti-hypertensive agents active as ouabain antagonists. The pathogenetic mechanisms through which increased EO levels affect cardiovascular system involve the modulation of Na-K ATPase, the key enzyme responsible for renal tubular sodium reabsorption and the activation of signalling transduction pathways implicated in growth-related gene transcription. By studying both genetic and experimental rat models of hypertension and comparing them with humans, our group has demonstrated that elevated levels of circulating EO and the genetic polymorphism of the cytoskeletal protein adducin associate with hypertension and high renal Na-K pump activity. Ouabain itself induces hypertension and up-regulates renal Na-K pump when chronically infused at low doses into rats (OS). In renal cultured cells, either incubated for several days with nanomolar concentrations of ouabain or transfected with the hypertensive adducin genetic variant, the Na-K pump results enhanced. Moreover, both EO and adducin polymorphism affect cardiac complications associated to hypertension, the former through the activation of a signalling transduction pathway. As a consequence, a compound able to interact with the cellular and molecular alterations, sustained by EO or mutated adducin, may represent the suitable treatment for those patients in whom these mechanisms are at work. A new antihypertensive compound, PST 2238, that selectively antagonises the pressor effect and the alteration of renal Na-K pump, sustained both by ouabain and adducin polymorphism, is described. A selective ability of PST 2238 to antagonise the ouabain-induced organ hypertrophy is also documented. The specificity of PST 2238 mechanism of action is supported by the absence of interactions with receptors or hormones involved in blood pressure regulation and by the lack of diuretic activity and diuretic-associated side effects. It is concluded that this compound could be useful for the treatment of those forms of essential hypertension in which renal Na handling alterations and cardiac complications are associated with either increased EO levels and/or adducin polymorphism.

Renal Na,K-ATPase in genetic hypertension.

Milan hypertensive rats (MHS) develop hypertension because of a primary renal alteration. Both apical and basolateral sodium transport are faster in membrane vesicles derived from renal tubules of MHS than in those of Milan normotensive control rats (MNS). These findings suggest that the increased renal sodium retention and concomitant development of hypertension in MHS may be linked to an altered transepithelial sodium transport. Since this transport is mainly under the control of the Na-K pump, we investigated whether an alteration of the enzymatic activity and/or protein expression of the renal Na,K-ATPase is detectable in prehypertensive MHS. We measured the Na,K-ATPase activity, Rb+ occlusion, turnover number, alpha 1- and beta 1-subunit protein abundance, and alpha 1 and beta 1 mRNA levels in microsomes from renal outer medulla of young (prehypertensive) and adult (hypertensive) MHS and in age-matched MNS. In both young and adult MHS, the Na,K-ATPase activity was significantly higher because of an enhanced number of active pump sites, as determined by Rb+ occlusion maximal binding. The higher number of pump sites was associated with a significant pretranslational increase of alpha 1 and beta 1 mRNA levels that preceded the development of hypertension in MHS. Since a molecular alteration of the cytoskeletal protein adducin is genetically associated with hypertension in MHS and is able to affect the actin-cytoskeleton and Na-K pump activity in transfected renal cells, we propose that the in vivo upregulation of Na-K pump in MHS is primary and linked to a genetic alteration of adducin.

Genomic organisation and chromosomal localisation of the gene encoding human beta adducin.

Adducin (ADD) is a heterodimeric protein of the membrane skeleton with subunits of 103 (alpha) and 97 kDa (beta). It promotes the assembly of the spectrin-actin network. We have previously shown that one point mutation in each of the alpha and beta rat ADD-encoding genes is associated with blood pressure variation in an animal model for hypertension, the Milan hypertensive strain of rats, probably due to a change in the phosphorylation pattern. In fact, the rat mutations, Y to F for alpha and R to Q for beta, are located, respectively, in a tyrosine kinase and a protein kinase A phosphorylation site. We have now determined, for the human beta-ADD-encoding gene, its chromosomal localisation, exon-intron organisation and alternative splicing patterns. We report here that human beta-ADD is localised on chromosome 2 and we also show a characteristic 3' end alternative splicing of the beta-ADD RNA that generates two distinct beta-ADD families, namely ADD 63 and 97; both of them in turn present a very complex differential splicing pattern in the internal exons.

Characterisation and chromosomal localisation of the rat alpha- and beta-adducin-encoding genes.

A polymorphism in the genes encoding alpha- and beta-adducin (ADD) was described as being associated with blood-pressure variation in a genetically hypertensive strain of rats (MHS). ADD is a cytoskeletal heterodimeric protein which may be involved in cellular signal transduction and interacts with other membrane skeleton proteins which affect ion transport across the cell membrane. The cDNA encoding the alpha subunit of rat ADD was isolated using PCR methods. The cDNA consists of about 3900 bp and encodes a protein of 735 amino acids (aa) which shows 91% aa identity with the human counterpart. In spleen and kidney, three alternative spliced exons were found by PCR amplification and confirmed by RNase protection analysis. 17 inbred rat strains were genotyped for the polymorphism in the alpha- and beta-ADD genes. Chromosomal localisation mapped rat alpha-ADD on chromosome 14 and rat beta-ADD on chromosome 4.

Calcium ATPase in erythrocytes of spontaneously hypertensive rats of the Milan strain.

Calcium (Ca)-dependent ATPase activity was determined in erythrocyte membrane ghosts from normal and spontaneously hypertensive rats of the Milan strain in order to detect changes in enzyme activity which had previously been shown in the spontaneously hypertensive rat strain. Activity was similar in control and hypertensive rats in the absence of calmodulin. In contrast, activity in the presence of saturating amounts of calmodulin was significantly lower in the hypertensive rats. At the Vmax (free Ca2+ concentration 10 mumol/l) the decrease was about 30% (70.1 +/- 8.94 versus 49.1 +/- 4.75 nmol of ATP split/mg of ghost proteins per min; P less than 0.05). The affinity of the ATP-dependent Ca2+ pump for Ca2+ (Km about 1 mumol/l) was not altered in the hypertensive rats. It is possible that deficient Ca ATPase activity sustains an increase of intracellular free Ca in cells of hypertensive rats concomitant with the intracellular sodium (Na) decrease typical of this strain.

Antihypertensive effect of captopril, canrenoate potassium, and atenolol. Relations with red blood cell sodium transport and renin.

We compared the antihypertensive efficacy of atenolol (100 mg/d), canrenoate potassium (200 mg/d), and captopril (75 mg/d) in 30 essential hypertensives. The three drugs were administered in a randomized change-over sequence for four-months each. The main variables associated with blood pressure regulation were measured in the basal condition and at the end of each treatment period. The erythrocyte Na transport systems were measured only in the basal condition and at the end of the first treatment period. The average blood pressure reduction was similar for each drug. Mean blood pressure levels after captopril correlated positively with those after atenolol in the individual patients (P less than 0.0001); mean blood pressure levels after canrenoate potassium, on the contrary, did not correlate with those after the other two drugs. Captopril and canrenoate potassium treatment reduced intraerythrocyte Na content (P less than 0.02), canrenoate potassium increased Na-K pump (P0.05), atenolol did not change any erythrocyte membrane Na transport parameters. The ouabain-resistant Na transport systems were not modified by any drug. The patients were divided in three groups according to their antihypertensive response: nonresponders (six patients), canrenoate potassium responders (nine patients) and captopril-atenolol responders (15 patients, equally responsive to both drugs). Nonresponders had the lowest basal Na pump (P less than 0.02). Canrenoate potassium responders had higher basal Na-K cotransport than captopril-atenolol responders (P less than 0.02). Atenolol-captopril responders had the highest basal plasma renin activity (PRA, P less than 0.02). The blood pressure reduction after atenolol correlated with the induced fall in PRA (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

A possible primary role for the kidney in essential hypertension.

The role of the kidney in "essential" or "genetic" types of hypertension has been evaluated both in a rat model such as the Milan hypertensive strain (MHS) or in humans. In both species, abnormalities of renal function have been demonstrated before the development of hypertension. Moreover hypertension could be "transplanted" with the kidney when kidney cross-transplantation was carried out between MHS and the Milan normotensive strain (MNS). Also in humans, the familiality for hypertension of the donor affected the requirement of antihypertensive therapy of the recipient. Further studies furnished results that were consistent with the hypothesis that a primary increase in Na transport across the tubular cell could be responsible for the pressor effect of the kidney in MHS or in humans. Because many similarities were found between the function of the tubular cell and the red blood cell in MHS, red blood cells were used to gain information about the molecular genetic mechanisms underlying these cellular changes. The results so far obtained showed that red blood cell abnormalities in MHS were genetically determined within the stem cells and genetically associated with the development of hypertension in F2 hybrids obtained by crossing the F1 (MHS X MNS). Moreover, the abnormal Na transport across the cell membrane may be due to an abnormal function of the membrane skeleton proteins. Studies are in progress to evaluate a possible cause-effect relationship between: membrane skeleton protein-ion transport across the cell membrane and the development of hypertension.

Heritability estimate of erythrocyte Na-K-Cl cotransport in normotensive and hypertensive families.

Na-K-Cl cotransport was measured in 209 essential hypertensive patients (EH) and in 114 normotensive controls (NT). The distribution of Na-K-Cl cotransport was bimodal in EH and unimodal in NT. The EH with higher Na-K-Cl cotransport values had increased passive permeability to Na in fresh erythrocytes and increased Li-Na countertransport compared to NT. Li-Na countertransport was significantly increased in the EH as a whole, but the increase was accounted for by some EH individuals with elevated Na-K-Cl cotransport values. A simple biometric analysis of the Na-K-Cl cotransport was performed for 287 individuals of 86 families with different prevalence of hypertension (neither parent hypertensive, 39 families; one, 31 families; or both, 16 families). Na-K-Cl cotransport was not correlated between spouses, but was correlated highly significantly between the average value of the two parents (mid-parent) and offspring. The polygenic additive heritability (h2) was about 50% for all families considered together. It increased slightly for the hypertensive families analyzed alone (no significant correlation was found, and hence genetic heritability, in the normotensive families). Finally, after applying arbitrary cut-off points to the Na-K-Cl cotransport values, segregation analysis showed that some major gene, recessive for the high allele, also contributes to the phenotypic value of Na-K-Cl cotransport.

Antihypertensive compounds that modulate the Na-K pump.

A primary impairment of the kidney sodium excretion has been documented both in hypertensive patients (EH) and genetic animal models (Milan hypertensive rat [MHS]) carrying mutations of the cytoskeletal protein adducin and/or increased plasma levels of endogenous ouabain (EO). Ouabain (OU) itself induces hypertension in rats and both OU and mutated adducin activate the renal Na/K-ATPase function both in vivo and in cultured renal cells (NRK). A new antihypertensive agent, PST 2238, able to selectively interact with these alterations has been developed. PST lowers blood pressure (BP) by normalizing the expression and activity of the renal Na-K pump selectively in those rat models carrying the adducin mutation (MHS) and/or increased EO levels (OS) at oral doses of 0.1-10 micro g/kg. In NRK cells either transfected with mutated adducin or incubated with 10(-9) M OU, PST normalizes the Na-K pump activity. Recently, an association between EO and cardiac complications has been observed in both EH and rat models consistent with a prohypertrophic activity of OU. OS rats showed a 10% increase of left ventricle and kidney weights as compared with controls, and PST 2238 (1 micro g/kg OS) prevented both ventricle and renal hypertrophy. This effect was associated with the ability of PST to antagonize the OU-dependent activation of growth-related genes, in the membrane subdomains of caveolae. In conclusion, PST is a new antihypertensive agent that may prevent cardiovascular complications associated with hypertension through the selective modulation of the Na-K pump function.

PST 2238: a new antihypertensive compound that modulates the Na-K pump 'in vivo' and 'in vitro'.

A primary renal alteration due to a genetic polymorphism of the cytoskeletal protein adducin associated with an up-regulation of the renal Na-K pump and increased levels of ouabainlike factor (OLF) has been identified as a possible causes of hypertension in Milan rats (MHS). This adducin polymorphism has also been found to be associated with hypertension and the blood pressure changes related to renal Na handling in humans and increased OLF levels have been found in a relevant portion of hypertensive patients. Increased activity and expression of the Na-K pump has also been observed under the following 'in vitro' and 'in vivo' conditions: rat renal cells transfected with the 'hypertensive' variant of adducin, as compared with normal cells; normal rat renal cells incubated for 5 days with 10(-9) M ouabain and normal rats made hypertensive by a chronic infusion of low doses of ouabain (OS rats). An up-regulation of the Na-K pump seems therefore to be a common biochemical alteration induced both by an adducin polymorphism and/or chronic exposure to low concentrations of ouabain (or OLF). A new antihypertensive compound, PST 2238, that selectively antagonizes the pressor effect and the alteration of the renal Na-K pump induced both by an adducin polymorphism and OLF, is described. The ability of PST 2238 to lower blood pressure and normalize the Na-K pump both in MHS and OS rats suggests that this compound could be useful in the treatment of those forms of essential hypertension in which renal Na-handling alterations are associated with either adducin polymorphisms and/or increased OLF levels.