Application of the Midi Parasep® SF technique for the detection of L1 larvae of Angiostrongylus cantonensis in faeces of infected rats.

We investigated parasitic zoonoses caused by protozoans and helminths in urban and peri-urban rat populations (Rattus norvegicus and R. rattus) in Spanish cities. Rats were trapped and then dissected to remove adult helminths, and the contents of the large intestine were retrieved for the study of parasitic forms. The Midi Parasep® solvent free (SF) technique was used to concentrate the parasites in the intestinal contents. Some of the rats studied (n = 8) were infected by the rat lungworm, Angiostongylus cantonensis, whose first stage larvae (L1) are shed in rat faeces. After the concentration technique, L1 larvae were found in the sediment of 6 of the 8 positive rats. The two negative sediment samples were due to the presence of either only adult females or, in addition to males, only young females in the lungs of the rats. In view of our results, Midi Parasep® SF turned out to be a simple, rapid, inexpensive, and sensitive method to detect nematode larvae, such as the L1 larvae of A. cantonensis (or A. costaricensis), in natural and experimentally infected rats.

Coping strategies and anxiety in caregivers of palliative cancer patients.

The study purpose was to determine the relationship between coping strategies and anxiety in primary family caregivers of palliative cancer patients. A cross-sectional study was carried out in a Pain and Palliative Care Unit in Spain. Data were collected through interviews from fifty primary family caregivers of palliative cancer patients. Main research variables were: (1) dependent variable: anxiety (subscale of anxiety from Goldberg's scale); (2) independent variable: coping (Brief COPE); (3) control variables: functional capacity and perceived burden. Analyses comprised descriptive statistics, correlation coefficients and multiple linear regression. Anxiety was present in the majority of caregivers surveyed (76%). Anxiety was related to the perception of perceived burden (ß = 0.42, P < 0.001), the emotion-focused coping (ß = -0.28, P = 0.01) and dysfunctional coping (ß = 0.41, P < 0.001), after adjusting for control variables. Thus, emotion-focused coping is negatively associated with anxiety, while dysfunctional coping is positively associated with anxiety. Problem-focused coping is not related to anxiety. Assessment of coping should be done in a systematic way in caregivers of palliative cancer patients.

Toxoplasma gondii detection and viability assays in ham legs and shoulders from experimentally infected pigs.

Epidemiological studies of toxoplasmosis show that infection in humans is mainly caused by the consumption of raw, undercooked or cured meat. Cured "Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. The "Serrano" ham is prepared from pork meat and undergoes a process known as curing and a subsequent fermentation without thermal or smoking treatments. The viability of Toxoplasma gondii in hams and shoulders from experimentally infected pigs that have been subject to different curing processes has been studied in order to evaluate the best method to completely eliminate the viable protozoa. The different treatments include, i) freezing the legs and shoulders below -20 °C for 3 days before salting with marine salt, ii) salting the meat with marine salt and nitrites, iii) salting only with marine salt (traditional process) and iv) salting with marine salt and then freezing at -20 °C for 3 days after the curing period. The ham leg samples were cured for 7 months and the shoulder samples for 5 months. The presence of T. gondii in the different treatments was studied by a "magnetic-capture" method for the isolation of T. gondii DNA and a quantitative real-time PCR to estimate the T. gondii burden in the ham legs and shoulders. The infectivity capacity of T. gondii in positive samples was assayed by bioassays in mice and some physicochemical parameters, such as pH, water activity (aw) and salt content, were evaluated at the end of the curing time. In all the cases where the samples were frozen the T. gondii infectivity was eliminated. In samples in which the meat was salted in marine salt plus nitrites, the parasite viability remained for longer than in the traditional salting process. The methods described here could be useful for producers to guarantee the safety of their products.

Quantification and viability assays of Toxoplasma gondii in commercial "Serrano" ham samples using magnetic capture real-time qPCR and bioassay techniques.

"Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. To make Serrano ham, pork undergoes a salting and a subsequent fermentation process known as curing. Certain pigs used for meat production are an important source of Toxoplasma gondii infection in humans. We have developed a method for quantifying and assaying the viability of the T. gondii present in commercial Serrano ham samples. A magnetic capture method for the isolation of T. gondii DNA and a qRT-PCR were used to estimate the T. gondii burden in 475 commercial samples of "Serrano" ham in two presentation formats: ham pieces and sliced ham. The infectivity capacity of T. gondii in positive samples was assayed in mice. The global prevalence of T. gondii was 8.84%, ranging from 32.35% in one of the companies to 0% prevalence in three other companies. The infectivity assays revealed that only 4.84% of the positive samples were infective. To the best of our knowledge this is the first report focussing on the prevalence of T. gondii in commercial "Serrano" ham. The method described here could be useful for producers to guarantee the safety of their products.

Function and expression of renal epithelial sodium transporters in rats with thyroid dysfunction.

Thyroid disorders are accompanied by major changes in renal sodium handling and blood pressure. Sodium transporters play a crucial role in regulating sodium excretion. We determined the function and expression of type 3 Na/H (NHE3) exchanger, type 2 Na+K+2Cl co-transporter (NKCC2) co-transporter, NaCl co-transporter (NCC) cotransporter, and epithelial sodium channel (ENaC) in hypoand hyperthyroid rats at 6 weeks after each thyroid disorder induction. We measured the renal response to functional blockade of the tubular sodium transporters, using acetazolamide to inhibit the activity of NHE3, furosemide for NKCC2, hydrochlorotiazide for NCC, and amiloride for ENaC. Expression of sodium transporters was analyzed by measuring the protein abundance by Western blot. The responsiveness to NHE3 inhibition and NHE3 protein was lower in hypothyroid rats and higher in hyperthyroid rats vs controls. Hypothyroid rats showed greater diuretic and natriuretic responses to NKCC2 and ENaC blockade and higher protein abundance of NKCC2 vs controls. Hyperthyroid rats showed greater protein expression of NKCC2 and NCC vs controls. Groups did not differ in responsiveness to NCC blockade. The expression and activity of ENaC were lower in hyperthyroid rats. In conclusion, reduced NHE3 activity may participate in the low blood pressure of hypothyroid rats and elevated NHE3 activity in the high blood pressure of hyperthyroid rats. These proximal alterations are counter-balanced by functional upregulation of NKCC2 and ENaC in downstream nephron segments of hypothyroid rats and by downregulation of αENaC activity and expression in hyperthyroid rats.

High mortality due to gastrointestinal TB in HIV and non-HIV patients.

BACKGROUND: Due to the reported low incidence of gastrointestinal TB, there is a lack of data related to the prognosis, risk factors and frequency of resistant TB in this subgroup of patients.OBJECTIVE: To report the clinical presentation, diagnostic methods, treatment and outcomes in gastrointestinal TB.METHODS: We prospectively studied the demographic, clinical, and paraclinical data of all consecutive gastrointestinal TB inpatients over an 8-year period.RESULTS: We identified gastrointestinal TB in 28 (3.5%) out of 799 inpatients with TB infection. Seven patients (25%) were HIV-positive. Overall mortality was 35.7%, with the combined variable of haemoglobin <12 g/dL and albumin <2.8 g/dL being independently associated with mortality (OR 25.7, 95% CI 1.405-471.1, P = 0.029). No difference in the need for surgery (28.6% vs. 47.6%, P = 0.662), occurrence of septic shock (14.3 vs. 23.8%, P = 1.00) or mortality (14.3% vs. 42.9%, P = 0.364) was found between HIV and non-HIV patients.CONCLUSION: Gastrointestinal TB was rare among TB patients in Hospital Universitario "Dr José E. González" (3.5%), but had a high mortality rate (35.7%). Clinical evolution, drug susceptibility patterns and outcomes were similar in HIV and non-HIV patients. In both groups, the combined haemoglobin and albumin variable on admission was clearly associated with mortality.

Efficacy and safety of conversion from twice-daily to once-daily tacrolimus in a large cohort of stable kidney transplant recipients.

Prolonged-release tacrolimus was developed to provide a more convenient once-daily dosing that could improve patient adherence. We conducted a multicenter, prospective, observational, 12-month study to describe the efficacy, safety and patient preference of conversion from tacrolimus twice-daily to once-daily formulation in stable kidney transplant recipients in routine clinical practice. Conversion was made on a 1 mg: 1 mg basis (1 mg: 1.1 mg in patients with trough levels <6 ng/mL). The study included 1832 patients (mean age (± SD): 50.0 ± 13.4 years; 62.7% male). After conversion, a modest reduction in tacrolimus trough levels, necessitating an increase in daily dose, was observed (mean changes at 12 months of -9.1% and +1.24%, respectively; p < 0.0001). Mean glomerular filtration rate did not change significantly (56.5 ± 19.7 mL/min at conversion vs. 55.7 ± 20.6 mL/min at 12 months). Proteinuria, blood pressure, lipid, hepatic and glucose parameters remained stable. Eight patients (0.4%) had acute rejection and 34 patients (1.85%) discontinued treatment. Almost all patients (99.4%) preferred the once-daily formulation, because of less frequent dosing (66%) and improved adherence (34%). In conclusion, at similar doses to twice-daily tacrolimus, once-daily formulation provided stable renal function, a low acute rejection rate, and good tolerability in stable kidney transplant recipients in the routine clinical practice setting.

Detection of Wolbachia bacteria in multiple organs and feces of the triatomine insect Rhodnius pallescens (Hemiptera, Reduviidae).

At least two types of Wolbachia bacteria were detected in wild and insectarium-raised Rhodnius pallescens, a natural vector of Trypanosoma cruzi and Trypanosoma rangeli. Wolbachia was detected in all the organs and tissues studied and in the feces, and this provided a methodological advantage for determining the presence of this endosymbiont in this host, obviating the need to kill the specimens. The occurrence of trypanosomatids in wild individuals was also studied.

Study of the stress proteins secreted by Leishmania donovani after treatment with edelfosine, mitelfosine and ilmofosine, and morphological alterations analyzed by electronic microscopy.

We studied the stress proteins induced in protozoa Leishmania donovani after treatment with edelfosine, miltefosine and ilmofosine. We studied the morphological and structural modifications caused in the promastigote forms of the parasite after treatment with the three alkyl-lysophospholipids (ALPs). A resistant strain of L. donovani to miltefosine was obtained and the morphological modifications were observed. The stress proteins induction was studied in promastigote forms and also in amastigote-like forms obtained in vitro. The proteins synthesized with the three alkyl-lysophospholipids were compared to those obtained by heat shock. The axenic amastigote forms synthesized a pattern of different proteins for those observed in the promastigote forms. The morphological alterations were observed under electronic microscopy. The membrane and mitochondria were the organs most affected by the three ALPs. We noted an apparition of vacuoles and vesicles in the treated promastigotes. In the resistant strain, we noted myelin bodies in the treated and untreated parasites.

Intestinal parasitism in the animals of the zoological garden "Peña Escrita" (Almuñecar, Spain).

Gastrointestinal parasites cause serious diarrhoea in captive animals. Therefore, we have undertaken this study to establish programmes to prevent, control, and treat intestinal parasitism in the animals of the zoological garden "Peña Escrita" of Almuñecar (Granada). An annual survey was conduced to estimate the occurrence of gastrointestinal parasites and the seasonality of this parasitism. Between June 2006 and May 2007, 432 samples were collected from primates, carnivores, perissoodactyla, artiodactyla, rodentia, diprotodontia, galliformes, anseriformes and struthioniformes. One or more intestinal parasites were identified in 72.5% of the animals. The most frequent pathogenic endoparasites were Eimeria spp. (17.3%), Trichuris spp. (5.1%), Strongyloides spp. (4.5%), Cyclospora spp. (4.5%), Cryptosporidium spp. (3.2%) and Isospora spp. (2.6%). Iodamoeba butschlii, Parascaris equorum and Trichuris spp. did not vary with season and Cryptosporidium spp., Dicrocoelium dendriticum, Metastrongylus spp. and Cylicospirura spp. appeared exclusively in Artiodactyla. Multiple parasitic infections were common, 70% of animals presented with at least two parasites (maximum=6). The most frequent cases of multiple parasitism were Eimeria spp. plus Blastocystis spp. and Eimeria spp. plus Nematodirus spp., in the last case the animals presented explosive diarrhoea. In accord with our results, after each sampling, some of the affected animals were treated and the corresponding programmes of prevention and control were designed.

Hepatitis C virus infection after renal transplantation.

Hepatitis C virus (HCV) infection is the main cause of liver disease after renal transplantation. Most patients have seroconverted on dialysis to positive RNA. The viral load increases during immunosuppressive therapy. The risk of developing chronic liver disease is related to the histopathologic findings, duration and severity of the disease, immunosuppression, and transplantation time. Hepatitis C virus infection can predict onset, of proteinuria and diabetes. We studied 868 patients who received renal transplants between (1987 and 2006), of whom 18.7% were seropositive for HCV. We observed a higher rate of HCV-seropositive patients related to the duration of hemodialysis therapy. Of the HCV seropositive patients, 77% had received renal allografts before 1998. There was no difference between the sexes; however, the HCV positive patients were younger. Polymerase chain reaction tests results were positive in 91.6% of the patients with HCV antibodies. The prevalence of diabetes was greater among HCV positive patients, as was as the persistence of proteinuria. Cryoglobulins were positive in 30.8%. The incidence of acute rejection episodes in the first year was similar between groups. Of the HCV-positive patients, 80.2% were treated with cyclosporine, most patients continued this therapy throughout the study. We observed no significant difference in mortality end graft survival rate between the two groups. However, renal function differed significantly at some points during the evolution of the clinical course. Renal transplantation is still the best treatment option in patients with chronic renal disease.

[Daclizumab in combination with mycophenolate mofetil and a late introduction of Tacrolimus at low doses, as a therapeutic approach in the elderly renal transplant donor-recipients pairs in kidney transplant]. / Daclizumab en combinación con Micofenolato Mofetil e introducción tardía de Tacrolimus a dosis bajas, como opción terapéutica en la pareja donante-receptor añoso en trasplante renal.

BACKGROUND: Nowadays, it is more frequent the use of kidneys from older donors in the renal transplantation. Moreover, it is also increasing the age of the recipients due to the ageing of the population treated with hemodialysis. This makes that recipients become older more commonly. This situation raises specific problems in the renal graft and in the recipient as well. In this manuscript we present the results of a multicenter study that analyzed an immunosuppressive strategy specifically designed to elderly renal transplant donor-recipients. METHODS: Patients > or =50 years were transplanted from donors > or =55 years. Immunosuppressive strategy consisted of daclizumab (2 doses of 1mg/Kg) in combination with steroids, mycophenolate mofetil (2g/daily during the first 45 days and then adjusted according to local practice) and Tacrolimus. Tacrolimus was introduced between 5 and 7 day post-transplantation, adjusting the predose levels between 4-8 ng/mL. Mean follow-up was 12 months. RESULTS: A total of 133 patients were included in the study. Mean age of recipients and donors was 61.3+/-6.2 years and 64.4+/-5.3, respectively. 42.9% of patients needed dialysis during the first week (median 4 days). Between first month and first year, serum creatinine improved from 2.0+/-1.0 mg/dl to 1.5+/-0.4 mg/dl. Similar improvements were observed when creatinine clearance (Cockroft-Gault) was calculated. The survival of patient and renal graft at 12 months was 97.7% and 96.1%, respectively. The acute rejection rate was 13.5%. Security profile was good, as expected. CONCLUSIONS: The Daclizumab and mycophenolate mofetil regimen with a late introduction of Tacrolimus at low doses is a good alternative in the elderly renal transplant recipients with a low immunologic risk.

Characterization and functionality of two members of the SPFH protein superfamily, prohibitin 1 and 2 in Leishmania major.

BACKGROUND: Leishmaniasis, a disease caused by parasites of the genus Leishmania, infects roughly 12 million people worldwide, with about two million new cases per year. Prohibitins (PHBs) are highly conserved proteins belonging to the stomatin-prohibitin flotillin-HflC/K (SPFH) protein superfamily. In this study, we examine the potential functions of two proteins of Leishmania major, PHB1 and PHB2, as well as how they might help protect the protozoan against oxidative stress. RESULTS: By immunolocalization in the parasite cells, PHB1 appeared in the mitochondria and plasma membrane, whereas PHB2 was grouped in the nucleus. When Leishmania cells were under oxidative stress, PHB1 migrates towards the plasma membrane and the paraxial rod, while PHB2 remained in the nucleus and near the kinetoplast. PHB1 presented higher mRNA levels than PHB2 in the amastigotes and the infective metacyclic forms. The mRNA expression of both prohibitins was affected by the presence of the Fe3+ ion. PHBs inhibited the Fenton reaction, where reactive oxygen species could nick DNA, implying that they play a crucial role in controlling oxidative stress. CONCLUSIONS: Here, we propose that PHBs may help to protect membranes and DNA against superoxide ions, thus enhancing the survival capacity of the protozoan by controlling the ROS within the phagosome of the macrophages where the parasite multiplies.

Intraperitoneal administration of the anti-IL-23 antibody prevents the establishment of intestinal nematodes in mice.

Previous studies have established that an increased Th-9 response creates a hostile environment for nematode parasites. Given that IL-23, a cytokine required for maintenance of the IL-17-secreting phenotype, has inhibitory effects on IL-9 production, we hypothesized that reducing circulating IL-23 by treatment with anti-IL-23 antibodies would reduce the establishment and development of parasitic intestinal nematodes. In this study, we show that animals treated with anti-IL-23 monoclonal antibodies showed a drastic reduction in the number of mouse pinworms (Aspiculuris tetraptera) recovered from the intestine (p < 0.001) at 23 days post-infection compared to the untreated animals. The cytokine levels in Peyer's patches (PP) in treated and infected animals increase the expression of interleukins such as IL-25, IL-21, and IL-9, augmenting mucus production in the crypts, and boosting chemokines, such as OX40 and CCL20 in the mucosa. Our results suggest that the Th17/Th2 regulatory mechanism provoked by the administration of the anti-IL-23 antibody prevents the implantation of the intestinal nematode in mice. The diminished inflammatory IL-17 levels alter the Th9 environment perhaps as a consequence of IL-17 inhibiting IL-9 expression. These Th9 conditions may explain the successful treatment against Inflammatory Bowel Disease (IBD) both with antibodies against IL-23 or through parasitization with nematodes.

Renal Transplant Immunosuppression in Patients With Hemolytic Uremic Syndrome: Four Case Reports.

A high rate of recurrence has been described in atypical hemolytic uremic syndrome renal transplant recipients, favored by certain immunosuppressant drugs that can induce complement activation. We present four case series in which three patients were diagnosed pretransplantation and a fourth who had onset in the very early post-transplantation period. The patients received different immunosuppression schedules, and all had improvement after more than 2-years. We suggest the need to stratify the risk of atypical hemolytic uremic syndrome recurrence using genetic studies and the available drugs as the main factors that allow graft survival improvement today.

Poly(ADP-ribose) polymerase expression in kidney transplantation: from alfa (alpha) to Omega (Omega).

INTRODUCTION: Overactivation of the enzyme poly(ADP-ribose) polymerase (PARP-1) can be induced by ischemia-reperfusion and involved in the renal injury subsequent to kidney transplant. The poly(ADP-ribosy)lation mechanism alters free radical-induced DNA damage, which is repair by PARP-1 polymer. However, PARP-1 overexpression induces cellular necrosis. Our aim was to study the immunohistochemical PARP-1 expression in kidney transplant biopsies associated with various events. MATERIALS AND METHODS: We studied the nuclear expression of PARP-1 in kidney tubule cells by immunohistochemistry using the monoclonal antibody PAR01 in donor biopsies without acute tubular necrosis (ATN) (n = 60; controls), allografts that suffer ATN (n = 90) or an episode of acute humoral rejection (n = 12) or acute tubulointerstitial rejection (n = 25), or chronic allograft nephropathy (n = 25). Furthermore, we also studied protocol biopsies with subclinical rejection (n = 60). Renal lesions in transplant biopsies were graded blindly using 1997 Banff criteria without any clinical information. RESULTS: Biopsies without morphological features of ATN, namely acute tubulointerstitial rejection, borderline or subclinical rejection, showed lesser PARP-1 expression compared with biopsies with ATN or with ischemic mechanism of acute humoral rejection or chronic allograft nephropathys. We observed an inverse relation between PARP-1 expression and renal function (P < .001). Overall, renal biopsies showing ATN revealed greater expression of PARP-1 (r = 0.785, Pearson test). A significant relationship with PARP-1 expression was demonstrated with renal function (effective diuresis, serum creatinine levels) and pretransplant cold ischemia time (P < .001). CONCLUSION: Kidney transplant events including ischemia were associated with the highest PARP-1 expression and worse allograft renal function.

Effect of alkyl-lysophospholipids on some aspects of the metabolism of Leishmania donovani.

Alkyl-lysophospholipids (ALPs), developed initially to be antitumor agents, have proved highly effective in the treatment of visceral leishmaniasis, a disease caused by the species making up the protozoan complex Leishmania donovani. Although their effectiveness is known, the mode of action against this parasite is not completely understood. In the present work, we have studied the effect of 3 derivatives, edelfosine, miltefosine, and ilmofosine. Using nuclear magnetic resonance spectroscopy ('H-NMR), we have examined the excreted catabolites from glucose metabolism in the promastigote forms treated with these compounds. The ALPs at concentrations of 19 and 38 microM inhibit the excretion of acetate, succinate, and pyruvate. The effect of edelfosine, miltefosine, and ilmofosine on the activity of the enzymes hexokinase, glycerolkinase 3-PD, phosphoglucose isomerase, superoxide dismutase, and phospholipase C were also examined. Glycerolkinase 3-PD and phosphoglucose isomerase are generally insensitive to the compounds, whereas hexokinase and superoxide dismutase are inhibited by miltefosine and ilmofosine. The ALPs exhibited an activated effect against the phospholipase C activity. Alkyl-lysophospholipids were shown to have a significant effect on several enzymes in important biochemical pathways indispensable for the survival of L. donovani promasigotes.

Self-adjuvanting C18 lipid vinil sulfone-PP2A vaccine: study of the induced immunomodulation against Trichuris muris infection.

Despite the importance of the adjuvant in the immunization process, very few adjuvants merge with the antigens in vaccines. A synthetic self-adjuvant oleic-vinyl sulfone (OVS) linked to the catalytic region of recombinant serine/threonine phosphatase 2A from the nematode Angiostrongylus costaricensis (rPP2A) was used for intranasal immunization in mice previously infected with Trichuris muris The animal intranasal immunization with rPP2A-OVS showed a reduction of 99.01% in the number of the nematode eggs and 97.90% in adult. The immunohistochemical analysis of the intestinal sections showed that in immunized animals with lipopeptide the mucus was significantly higher than in the other experimental groups. Also, these animals presented significantly different chemokine, CCL20 and CCL11, levels. However, although the number and size of Tuft cells did not vary between groups, the intensity of fluorescence per cell was significant in the group immunized with the rPP2A-OVS. The results of the present study suggest that mice immunized with the lipopeptide are capable of activating a combined Th17/Th9 response. This strategy of immunization may be of great applicability not only in immunotherapy and immunoprophylaxis to control diseases caused by nematodes but also in pathologies necessitating action at the level of the Th9 response in the intestinal mucosa.

Effect of statin treatment on oxidative stress and renal function in renal transplantation.

The beneficial action of statins on the lipid profile, cardiovascular disease, and death is well known. Besides their lipid-lowering role, these drugs have pleiotropic action that derive from their prevention of the synthesis of isoprenoids, mediators in cell signaling. Thus, due to their antioxidant capacity, statins can decrease the production of reactive oxygen species by inhibiting NAD(P)H oxidase activity. Previous studies by our group have described increased oxidative stress status in renal transplantation that might benefit from HMG CoA reductase inhibitor therapy. The aim of this study was to assess the influence of statins on stress parameters and their relevance to renal function in kidney transplantation. In 58 transplanted patients a first blood sample was obtained, without statins followed by 21 patients treated with statins (group 1) and 37 patients without drug (group 0) for a 6-month study period. We collected clinical data as well biochemical results on lipid profile, creatinine and oxidative stress. Lipid profile reduction was significant among group 1 compared with group 0. An increased glutathione peroxidase (GPx) among observed in all patients was greater in the statin-treated group (P = .006). No differences in creatinine or Cockroft-Gault values were observed between before versus after drug administration. In conclusion, statin treatment in renal transplantation improves the lipid profile and may increase GPx-measured antioxidant capacity but appears to have no short-term effect on renal function.

Aspects of the cytological activity of edelfosine, miltefosine, and ilmofosine in Leishmania donovani.

To discover the mode of action of alkyl-lysophospholipids in Leishmania donovani, we studied the effects of edelfosine, miltefosine, and ilmofosine on intracellular pH, the parasite's cell cycle, and the induction of apoptosis. The effect of the alkyl-lysophospholipids was combined with that of inhibitors of some pumps and exchange regulators of intracellular pH (Na+/ H+; Cl-/CO- 3; and the Na+/K+ ATPase). The effect of the 3 alkyl-lysophospholipids on intracellular pH was indirect; the primary action occurred in the parasite's cell membrane. To determine intracellular pH, we used flow cytometry for the macrophages and axenic amastigotes and spectrofluorometry for the promastigote forms. Apoptosis and the cell cycle were studied by flow cytometry. Treatment of the extracellular promastigote form of L. donovani with the 3 alkyl-lysophospholipids induced death by apoptosis, whereas in the infected cell they caused necrosis rather than apoptosis. Miltefosine and ilmofosine at doses of 38 microM caused G2/M cell cycle inhibition in L. donovani promastigotes.