[Detection of RAS genes mutation using the Cobas® method in a private laboratory of pathology: Medical and economical study in comparison to a public platform of molecular biology of cancer]. / Analyse des mutations des gènes RAS par technique Cobas® au sein d'une structure de pathologie libérale : étude médico-économique et moléculaire comparative avec une plateforme labellisée INCa.

In France, determination of the mutation status of RAS genes for predictive response to anti-EGFR targeted treatments is carried out by public platforms of molecular biology of cancer created by the French National Cancer Institute. This study aims to demonstrate the feasibility of these analyses by a private pathology laboratory (MEDIPATH) as per the requirements of accreditation. We retrospectively studied the mutation status of KRAS and NRAS genes in 163 cases of colorectal metastatic cancer using the Cobas® technique. We compared our results to those prospectively obtained through pyrosequencing and allelic discrimination by the genetic laboratory of solid tumors at the Nice University Hospital (PACA-EST regional platform). The results of both series were identical: 98.7% positive correlation; negative correlation of 93.1%; overall correlation of 95.7% (Kappa=0.92). This study demonstrates the feasibility of molecular analysis in a private pathology laboratory. As this practice requires a high level of guarantee, its accreditation, according to the NF-EN-ISO15189 quality compliance French standard, is essential. Conducting molecular analysis in this context avoids the steps of routing the sample and the result between the pathology laboratory and the platform, which reduces the overall time of rendering the result. In conclusion, the transfer of some analysis from these platforms to private pathology laboratories would allow the platforms to be discharged from a part of routine testing and therefore concentrate their efforts to the development of new analyses constantly required to access personalized medicine.

Association of Oncogenic Mutations in Patients With Advanced Cutaneous Squamous Cell Carcinomas Treated With Cetuximab.

IMPORTANCE: Cetuximab was recently proposed for advanced cutaneous squamous cell carcinomas (cSCC); however, its efficacy is inconsistent and identification of predictive biomarkers for response is necessary. OBJECTIVE: To search for somatic mutations of the HRAS, KRAS, NRAS, BRAF, and EGFR genes in patients with advanced cSCC treated with cetuximab; and to investigate the efficacy and tolerance of cetuximab according to these mutations. DESIGN, SETTING, AND PARTICIPANTS: A multicentric and retrospective study of 31 patients (22 men, 9 women) with histologically confirmed advanced cSCC carried out in 1 department of dermatology and 2 departments of medical oncology in France between January 2008 and December 2014. The median age of participants was 86 years (range, 48-96 years). INTERVENTIONS: Mutational status was determined by pyrosequencing method, allelic discrimination, or Sanger sequencing. Patients were treated by single-agent cetuximab. MAIN OUTCOMES AND MEASURES: The primary end point was the incidence of somatic mutations of the RAS, BRAF, and EGFR genes and association of cetuximab efficacy with these mutations was investigated by using Fisher test. Secondary end points were the disease control rate (DCR) at week 6, the progression free-survival (PFS), overall survival (OS), and safety profile of cetuximab. RESULTS: Thirty-one samples of cSCC from 31 patients were analyzed. Only 2 RAS mutated samples (6.5%) were identified. The first harbored a NRAS point mutation (c.35G>A) in codon 12, resulting in a p.G12D substitution. The second sample presented a HRAS point mutation (c.38G>T) in codon 13, resulting in a p.G13V substitution. No mutation of KRAS, BRAF, and EGFR genes at the investigated loci was found. Two patients with NRAS and HRAS mutations showed a partial and complete response to cetuximab, respectively. The mean duration of follow-up was 19 months. At week 6, the disease control rate was 67.8%. The median OS was 13 months and the median PFS was 9 months. All patients could continue cetuximab treatment without dose reduction. CONCLUSIONS AND RELEVANCE: Even in elderly patients with advanced cSCC, cetuximab was efficacious and well-tolerated. This suggests that cetuximab is certainly warranted in the treatment of advanced cSCC. However, it is also important to identify tumor specific mutations that may determine response to treatment and prognosis for the disease. We have identified here that the incidence of RAS, BRAF, and EGFR mutations is low in cSCC.

Peritoneal carcinomatosis of colorectal cancer: novel clinical and molecular outcomes.

BACKGROUND: The objective of this study was to identify the prognostic impact of parameters in peritoneal carcinomatosis from colorectal cancer. METHODS: We collected data from patients treated by cytoreductive surgery and Hyperthermic Intraperitoneal Chemotherapy for peritoneal carcinomatosis secondary to colorectal cancer. RESULTS: Ninety-one procedures were performed. In univariate analysis, an increased peritoneal cancer index was associated with decreased survival (P < .001). The presence of signet ring cells was associated to a decrease in survival from 45.8 to 12.1 months (P < .001). Microsatellite sequences instability status was the only molecular prognostic factor correlated with an increase in median disease-free survival: 12.4 vs 24.9 months (P = .01). The presence of a mucinous component was associated with a decreased of survival from 51.9 to 35.1 months (P = .02). CONCLUSIONS: Clinical factors were affecting the survival of patients. The absence of signet ring cells and mucinous component and the presence of microsatellite sequences instability may be favorable prognostic factors.

Challenging a dogma: co-mutations exist in MAPK pathway genes in colorectal cancer.

Sequencing of genes encoding mitogen-activated protein kinase (MAPK) pathway proteins in colorectal cancer (CRC) has established as dogma that of the genes in a pathway only a single one is ever mutated. We searched for cases with a mutation in more than one MAPK pathway gene (co-mutations). Tumor tissue samples of all patients presenting with CRC, and referred between 01/01/2008 and 01/06/2015 to three French cancer centers for determination of mutation status of RAS/RAF+/-PIK3CA, were retrospectively screened for co-mutations using Sanger sequencing or next-generation sequencing. We found that of 1791 colorectal patients with mutations in the MAPK pathway, 20 had a co-mutation, 8 of KRAS/NRAS, and some even with a third mutation. More than half of the mutations were in codons 12 and 13. We also found 3 cases with a co-mutation of NRAS/BRAF and 9 with a co-mutation of KRAS/BRAF. In 2 patients with a co-mutation of KRAS/NRAS, the co-mutation existed in the primary as well as in a metastasis, which suggests that co-mutations occur early during carcinogenesis and are maintained when a tumor disseminates. We conclude that co-mutations exist in the MAPK genes but with low frequency and as yet with unknown outcome implications.

Three Rounds of External Quality Assessment in France to Evaluate the Performance of 28 Platforms for Multiparametric Molecular Testing in Metastatic Colorectal and Non-Small Cell Lung Cancer.

Personalized medicine has gained increasing importance in clinical oncology, and several clinically important biomarkers are implemented in routine practice. In an effort to guarantee high quality of molecular testing in France, three subsequent external quality assessment rounds were organized at the initiative of the National Cancer Institute between 2012 and 2014. The schemes included clinically relevant biomarkers for metastatic colorectal (KRAS, NRAS, BRAF, PIK3CA, microsatellite instability) and non-small cell lung cancer (EGFR, KRAS, BRAF, PIK3CA, ERBB2), and they represent the first multigene/multicancer studies throughout Europe. In total, 56 laboratories coordinated by 28 regional molecular centers participated in the schemes. Laboratories received formalin-fixed, paraffin-embedded samples and were asked to use routine methods for molecular testing to predict patient response to targeted therapies. They were encouraged to return results within 14 calendar days after sample receipt. Both genotyping and reporting were evaluated separately. During the three external quality assessment rounds, mean genotype scores were all above the preset standard of 90% for all biomarkers. Participants were mainly challenged in case of rare insertions or deletions. Assessment of the written reports showed substantial progress between the external quality assessment schemes on multiple criteria. Several essential elements such as the clinical interpretation of test results and the reason for testing still require improvement by continued external quality assessment education.

Spatio-temporal genetic heterogeneity of CTNNB1 mutations in sporadic desmoid type fibromatosis lesions.

Desmoid type fibromatosis (DT) is a rare lesion of unclear pathogenesis that most often presents a mutation of the (ß-catenin) gene. The natural history and clinical evolution are highly variable between patients and to date there is no consensus on optimal therapy. We report two cases of a patient with multiple DT lesions. Molecular investigations performed in both patients on multiple tumors at different anatomical sites revealed non-identical CTNNB1 mutations. The first patient was a 39-year-old man with a history of recurrent DT. In two of the DT lesions, three different mutations were found in codons 41 and 45, respectively. The lesions showed marked inflammatory features, characterized by IgG4 positive lymphoplasmacytic infiltrates and a foreign body reaction, which increased in intensity over time. The patient was eventually treated with a COX-2 inhibitor and the remaining mass was stabilized. In the two DT lesions of the second patient, CTNNB1 mutations S45P and T41A were found. The presence of different mutations in multiple focally recurrent sporadic DT lesions indicates that they do not have a clonal relationship. Our data suggest that a CTNNB1 mutation is a necessary event probably by providing a selective growth advantage. An IgG4 host antigen response is discussed as a potential predisposing factor for one of the patients.

EGFR, KRAS, BRAF, and HER-2 molecular status in brain metastases from 77 NSCLC patients.

The aim of this study was to determine the frequency of EGFR, KRAS, BRAF, and HER-2 mutations in brain metastases from non-small cell lung carcinomas (BM-NSCLC). A total of 77 samples of BM-NSCLC were included and 19 samples of BM from breast, kidney, and colorectal tumors were also studied as controls. These samples were collected from patients followed between 2008 and 2011 at Poitiers and Nice University Hospitals in France. The frequencies of EGFR, KRAS, BRAF, and HER-2 mutations in BM-NSCLC were 2.6, 38.5, 0, and 0% respectively. The incidence of KRAS mutation was significantly higher in female and younger patients (P < 0.05). No mutations of the four genes were found in BM from breast or kidney. However, among six BM from colorectal tumors, we identified KRAS mutations in three cases and BRAF mutations in two other cases. This study is the largest analysis on genetic alterations in BM-NSCLC performed to date. Our results suggest a low frequency of EGFR mutations in BM-NSCLC whereas KRAS mutations are as frequent in BM-NSCLC as in primitive NSCLC. These results raise the question of the variability of the brain metastatic potential of NSCLC cells in relation to the mutation pattern.

A renal metanephric adenoma showing both a 2p16e24 deletion and BRAF V600E mutation: a synergistic role for a tumor suppressor gene on chromosome 2p and BRAF activation?

Metanephric adenomas (MAs) are rare benign tumors that may be difficult to recognize. Specific genetic anomalies might aid in diagnosis, but genomic data are limited and conflicting. Consistent mutations of the BRAF gene have been recently reported in MAs and could become useful as a discriminative marker among renal tumors. We report here a case of MA, showing both a BRAF V600E mutation and a segmental loss within bands 2p16 and 2p24 as the sole quantitative genomic anomaly. We compared the borders and size of the deleted region in our case to those of five cases of MAs previously reported. We identified a common minimal region containing 87 genes, among which several tumor suppressor genes could be candidate actors in the pathogenesis of MA. We ruled out MSH2 and MSH6 as target gene candidates, both located in the deleted region, on the basis of preserved expression and microsatellite sequence stability. Our study confirms the recurrence of a BRAF mutation and of 2p alterations in MAs. This first case showing simultaneous presence of a BRAF mutation and a 2p deletion raises the question of a synergistic role for these two anomalies in the pathogenesis of MAs.

A newly characterized human well-differentiated liposarcoma cell line contains amplifications of the 12q12-21 and 10p11-14 regions.

While surgery is the usual treatment for localized well-differentiated and dedifferentiated liposarcomas (WDLPS/DDLPS), the therapeutic options for patients with advanced disease are limited. The classical antimitotic treatments are most often inefficient. The establishment of genetically characterized cell lines is therefore crucial for providing in vitro models for novel targeted therapies. We have used spectral karyotyping, fluorescence in situ hybridization with whole chromosome painting and locus-specific probes, and array-comparative genomic hybridization to identify the chromosomal and molecular alterations of a novel cell line established from a recurring sclerosing WDLPS. The karyotype was hypertriploid and showed multiple structural anomalies. All cells retained the presence of a giant marker chromosome that had been previously identified in the primary cell cultures. This giant chromosome contained high-level amplification of chromosomal regions 12q13-21 and lacked the alpha-satellite centromeric sequences associated with WDLPS/DDLPS. The 12q amplicon was large, containing 370 amplified genes. The DNA copy number ranged from 3 to 57. The highest levels of amplification were observed at 12q14.3 for GNS, WIF1, and HMGA2. We analyzed the mRNA expression status by real-time reverse transcription polymerase chain reaction for six genes from this amplicon: MDM2, HMGA2, CDK4, TSPAN31, WIF1, and YEATS4. mRNA overexpression was correlated with genomic amplification. A second amplicon originating from 10p11-14 was also present in the giant marker chromosome. The 10p amplicon contained 62 genes, including oncogenes such as MLLT10, previously described in chimeric fusion with MLL in leukemias, NEBL, and BMI1.

Accelerated clearance of Plasmodium-infected erythrocytes in sickle cell trait and annexin-A7 deficiency.

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE(2) formation, Ca(2+) permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7(-/-)) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7(-/-) mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7(-/-) mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE(2)-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stage-infected HbA/S or annexin-A7(-/-) erythrocytes and thus confers partial protection against malaria in vivo.

A high specificity and affinity interaction with serum albumin stimulates an anion conductance in malaria-infected erythrocytes.

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl(-) and lactate outward currents with an EC(50) of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

Purinoceptors are involved in the induction of an osmolyte permeability in malaria-infected and oxidized human erythrocytes.

In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stress induces a new organic osmolyte and anion permeability. To examine a role for autocrine purinoceptor signaling during this induction process, erythrocytic purinoceptor expression, and ATP release were determined. Furthermore, using pharmacological and genetic approaches the dependence on purinoceptor signaling of osmolyte permeability and Plasmodium development, both in vitro and in vivo, were assessed. Extracellular ATP did not induce an osmolyte permeability in non-infected or non-oxidized erythrocytes. ATP and other purinoceptor agonists increased the induction of osmolyte permeability during infection or oxidation as measured by isosmotic hemolysis and patch-clamp recording. Purinoceptor antagonists and apyrase decreased the induced permeability. The observed pharmacology suggested the involvement of P2Y purinoceptors. Accordingly, human erythrocytes expressed P2Y1 protein. Moreover, P2Y1-deficient mouse erythrocytes exhibited a delayed appearance of the osmolyte permeability during P. berghei infection- or oxidation compared with wild-type erythrocytes. Furthermore, the nonspecific purinoceptor antagonist suramin decreased in vitro growth and DNA/RNA amplification of P. falciparum in human erythrocytes and decreased in vivo growth of P. berghei. P. berghei developed slower in P2Y1-deficient mice in vivo compared with wild-type animals. In conclusion, induction of the osmolyte permeability in Plasmodium-infected erythrocytes involves autocrine purinoceptor signaling.

Permselectivity and pH-dependence of Plasmodium falciparum-induced anion currents in human erythrocytes.

Intraerythrocytic survival of the malaria pathogen Plasmodium falciparum requires delivery of nutrients and disposal of waste products across the host erythrocyte membrane. Recent patch-clamp experiments have demonstrated inwardly and outwardly rectifying anion conductances in infected but not in control erythrocytes. A ClC-2-generated fraction of the inwardly rectifying current is activated by cell swelling and presumably subserves host cell volume regulation. In contrast, the outwardly rectifying current is insensitive to cell volume but allows the passage of lactate and is involved in the transport of nutrients. The present study was performed to characterize the permselectivity and pH sensitivity of the anion conductances using whole-cell recording. The outwardly rectifying and the inwardly rectifying currents exhibited permselectivities of Cl- > or = Br- approximately I- > SCN- and SCN- > I- > Br- > Cl-, respectively, as evident from the reversal potentials recorded under biionic conditions. While the inwardly rectifying current was not affected significantly by alterations of pH between 6.0 and 8.4, the outward rectifier was inhibited strongly by alkalinization to pH > or = 7.8. Fluxes of 14C-lactate and parasite growth were decreased markedly by the increase of bath pH, an effect that may at least in part be due to inhibition of the outward rectifier and subsequently impaired transport across the erythrocyte membrane.

Stimulation of erythrocyte ceramide formation by platelet-activating factor.

Osmotic erythrocyte shrinkage leads to activation of cation channels with subsequent Ca2+ entry and stimulates a sphingomyelinase with subsequent formation of ceramide. Ca2+ and ceramide then activate a scramblase leading to breakdown of phosphatidylserine asymmetry of the cell membrane. The mediators accounting for activation of erythrocyte sphingomyelinase and phosphatidylserine exposure remained elusive. The study demonstrates that platelet-activating factor (PAF) is released from erythrocytes upon hyperosmotic cell shrinkage. The experiments further disclose the presence of PAF receptors in erythrocytes and show that PAF stimulates the breakdown of sphingomyelin and the release of ceramide from erythrocytes at isotonic conditions. PAF further triggers cell shrinkage (decrease of forward scatter) and phosphatidylserine exposure (annexin binding) of erythrocytes. The stimulation of annexin-binding is blunted by a genetic knockout of PAF receptors, by the PAF receptor antagonist ABT491 or by inhibition of sphingomyelinase with urea. In conclusion, PAF activates an erythrocyte sphingomyelinase and the then formed ceramide leads to the activation of scramblase with subsequent phosphatidylserine exposure.

Artemisinin inhibits cation currents in malaria-infected human erythrocytes.

BACKGROUND: Previous patch-clamp studies have demonstrated inwardly and outwardly rectifying anion currents, ClC-2 Cl- currents, and nonselective Ca(++)-permeable cation currents in Plasmodium falciparum-infected human erythrocytes. METHODS: The current work studied the effect of the potent antimalarial drug artemisinin on the P falciparum infection-induced whole cell currents in human erythrocyte. RESULTS: Artemisinin had no significant effect on the outwardly rectifying anion currents but inhibited the cation-selective currents with an apparent half-maximal inhibitory concentration of < or =10 micromol/L. CONCLUSION: Because artemisinin reportedly inhibits the asexual parasite amplification with much higher potency, the antimalarial action of the drug cannot be attributed to the artemisinin effect on the cation currents. However, artemisinin may be used as a pharmacologic tool to dissect different current fractions in P falciparum-infected erythrocytes.

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte.

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

Inhibition of erythrocyte phosphatidylserine exposure by urea and Cl-.

Osmotic shock by addition of sucrose to the medium stimulates erythrocyte sphingomyelinase with subsequent ceramide formation and triggers Ca(2+) entry through stimulation of cation channels. Both ceramide and Ca(2+) activate an erythrocyte scramblase, leading to breakdown of phosphatidylserine asymmetry, a typical feature of apoptosis. Because erythrocytes are regularly exposed to osmotic shock during passage of kidney medulla, the present study explored the influence of NaCl and urea on erythrocyte phosphatidylserine exposure as determined by annexin binding. The percentage of annexin-binding erythrocytes increased from <5 to 80 +/- 4% (n = 4) upon addition of 650 mM sucrose, an effect paralleled by activation of the cation channel and stimulation of ceramide formation. The number of annexin-binding erythrocytes increased only to 18% after addition of 325 mM NaCl and was not increased by addition of 650 mM urea. According to whole cell patch-clamp experiments, the cation conductance was activated by replacement of extracellular Cl(-) with gluconate at isotonic conditions or by addition of hypertonic sucrose or urea. Although stimulating the cation conductance, urea abrogated the annexin binding and concomitant increase of ceramide levels induced by osmotic cell shrinkage. In vitro sphingomyelinase assays demonstrated a direct inhibitory effect of urea on sphingomyelinase activity. Urea did not significantly interfere with annexin binding after addition of ceramide. In conclusion, both Cl(-) and urea blunt erythrocyte phosphatidylserine exposure after osmotic shock. Whereas Cl(-) is effective through inhibition of the cation conductance, urea exerts its effect through inhibition of sphingomyelinase, thus blunting formation of ceramide.

Enhanced susceptibility to erythrocyte "apoptosis" following phosphate depletion.

Among the sequelae of phosphate depletion is anaemia, due in part to a decreased life span of mature erythrocytes. Recent studies have disclosed that cellular stress leads to an increase of cytosolic Ca(2+) activity in erythrocytes thereby triggering cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane, both typical features of apoptosis. In the present experiments, phosphatidylserine exposure and cell size were measured by fluorescence-activated cell sorting (FACS) analysis of annexin binding and forward scatter, respectively. Erythrocytes from intact mice were compared with erythrocytes from mice exposed to a low-phosphate diet for 4 days. Annexin binding of freshly drawn erythrocytes was slightly but significantly enhanced by the low-phosphate diet. Furthermore, intracellular phosphate and ATP concentrations were significantly decreased in those erythrocytes whereas intracellular Ca(2+) activity was unaltered. Osmotic shock (exposure to 700 mOsm by addition of sucrose for 12 h), removal of Cl(-) (replaced by gluconate for 15 h) or removal of glucose (12 h) decreased cell volume and increased the number of annexin-binding erythrocytes. Interestingly, these effects were significantly larger in erythrocytes from phosphate-depleted animals. The experiments reveal a novel mechanism triggered by phosphate depletion that presumably contributes to the enhanced vulnerability and accelerated sequestration of erythrocytes and, thus, to anaemia.

Channel-induced apoptosis of infected host cells-the case of malaria.

Infection of erythrocytes by the malaria pathogen Plasmodium falciparum leads to activation of several distinct anion channels and a non-selective, Ca2+-permeable cation channel. All channel types are presumably activated by the oxidative stress generated by the pathogen. Similar or identical channels are activated by oxidation of non-infected erythrocytes. Activation of the non-selective cation channel allows entry of Ca2+ and Na+, both of which are required for intracellular growth of the pathogen. The entry of Ca2+ stimulates an intraerythrocytic scramblase that facilitates bi-directional phospholipid migration across the bilayer, resulting in breakdown of the phosphatidylserine asymmetry of the cell membrane. The exposure of phosphatidylserine at the outer surface of the cell membrane is presumably followed by binding to phosphatidylserine receptors on macrophages and subsequent phagocytosis of the affected erythrocyte. The lysosomal degradation may eventually eliminate the pathogen. The channel may thus play a dual role in pathogen survival. Absence of the channels is not compatible with pathogen growth, enhanced channel activity accelerates erythrocyte "apoptosis" that may represent a host defence mechanism serving to eliminate infected erythrocytes.

Organic osmolyte permeabilities of the malaria-induced anion conductances in human erythrocytes.

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new permeability pathways (NPPs) in the host cell membrane. Isotopic flux measurements demonstrated that the NPP are permeable to a wide variety of molecules, thus allowing uptake of nutrients and release of waste products. Recent patch-clamp recordings demonstrated the infection-induced up-regulation of an inwardly and an outwardly rectifying Cl(-) conductance. The present experiments have been performed to explore the sensitivity to cell volume and the organic osmolyte permeability of the two conductances. It is shown that the outward rectifier has a high relative lactate permeability (P(lactate)/P(Cl) = 0.4). Sucrose inhibited the outward-rectifier and abolished the infection-induced hemolysis in isosmotic sorbitol solution but had no or little effect on the inward-rectifier. Furosemide and NPPB blocked the outward-rectifying lactate current and the sorbitol hemolysis with IC(50)s in the range of 0.1 and 1 microM, respectively. In contrast, the IC(50)s of NPPB and furosemide for the inward-rectifying current were >10 microM. Osmotic cell-shrinkage inhibited the inwardly but not the outwardly rectifying conductance. In conclusion, the parasite-induced outwardly-rectifying anion conductance allows permeation of lactate and neutral carbohydrates, whereas the inward rectifier seems largely impermeable to organic solutes. All together, these data should help to resolve ongoing controversy regarding the number of unique channels that exist in P. falciparum-infected erythrocytes.