Serum level of vascular endothelial growth factor is influenced by erythropoietin treatment in peritoneal dialysis patients. (Grupo de Estudios Peritoneales de Madrid).

Some patients on long-term peritoneal dialysis (PD) develop a hyperpermeability state, owing to peritoneal neoangiogenesis. Vascular endothelial growth factor (VEGF), a potent mitogen for endothelial cells, has been implicated in most diseases characterized by microvascular neoformation. Erythropoietin (EPO) is able to induce endothelial proliferation in vitro. Our aim was to elucidate whether VEGF serum levels are influenced by EPO treatment, and whether VEGF serum level maintains a relationship with peritoneal transport data. We analyzed serum levels of VEGF in 35 PD patients (18 males, 17 females). Mean age was 58 years, with a mean time on PD of 98 +/- 75 months. Of the 35 patients, 19 were on automated peritoneal dialysis, and 16 were on continuous ambulatory peritoneal dialysis. Seven patients had diabetes. Peritoneal transport parameters were: urea mass transfer coefficient (MTC), 19.5 +/- 6.6 mL/min; creatinine MTC, 9.9 +/- 4.7 mL/min; net ultrafiltration, 491 +/- 166 mL per 4-hour dwell. Twenty seven patients were under therapy with recombinant human erythropoietin (rHuEPO). Mean serum VEGF levels were 347 +/- 203 pg/mL (range 66-857 pg/mL), with most patients in the normal range (60-700 pg/mL). VEGF levels did not correlate with age, sex, primary renal disease, diabetes, type of PD, time on PD, peritonitis, and cumulative glucose load. We found no correlation with urea MTC, creatinine MTC, ultrafiltration rate, or protein effluent levels. However, a significant negative correlation with residual renal function was seen (r = -0.39, p < 0.05). Patients treated with rHuEPO showed significantly higher serum levels of VEGF than non treated patients (375 +/- 220 pg/mL vs 251 +/- 75 pg/mL, p < 0.05), although they had similar residual renal function. We conclude that increased serum VEGF levels are associated with EPO treatment. Consequently, VEGF might have a role in the EPO effects found in PD patients. Whether both agents are related to peritoneal neoangiogenesis requires further research.

[Methods to assess the ex vivo growth capacity of mesothelial cells obtained directly from peritoneal effluent]. / Metodología para evaluar la capacidad de crecimiento ex vivo de las células mesoteliales obtenidas directamente del efluente peritoneal.

The anatomical and functional integrity of mesothelial cells (MC) is necessary for peritoneal membrane stability. At present, there is no satisfactory method to assess MC function and regenerative capacity in individual peritoneal dialysis (PD) patients. MC may be cultured from peritoneal biopsy specimens, but peritoneal biopsy is an invasive procedure that cannot be performed serially. The aim of this study is to explore the feasibility of serial culture of MC from the peritoneal effluent of PD patients. Fifty-two randomly selected PD patients were studied. MC were obtained from the peritoneal effluent of nocturnal 2.27% glucose exchanges and cultured in T25 tissue culture flasks. Subconfluent MC cultures were obtained in 80.7% of patients. At this stage, the percentage of cells in the tissue cultured flask characterized as MC by morphology and immunostaining had increased to 95.5%. MC were then subcultured in multi-well culture plates, where they showed exponential cell growth until day 16. Nine (17%) patients released low numbers of MC into the effluent and MC could not be cultured to subconfluence. One additional patient released and apparently adequate number of MC that repeatedly failed to reach confluence. Patients showed the same behavior in several cultures performed. In conclusion, peritoneal MC released into peritoneal effluent are accessible for profound analysis by a culture technique. This technique opens the possibility of serial follow-up of the biology of MC individual PD patients.

Metodología para evaluar la capacidad de crecimiento ex vivo de las células mesoteliales obtenidas directamente del efluente peritoneal / Ex-vivo growth capacity of human mesothelial cells obtained directly from peritoneal effluent

La integridad anatómica y funcional de la célula mesotelial (CM) es fundamental para la estabilidad de la membrana peritoneal. En la actualidad, no existe un método que permita comprobar la función y capacidad regenerativa de las CM de forma individual en los pacientes en diálisis peritoneal (DP). La CM puede ser cultivada a partir de muestras obtenidas de biopsia peritoneal, sin embargo es un procedimiento invasivo que no permite realizarse de manera repetida. El objetivo de este estudio es explorar la posibilidad de cultivar de forma reiterada las CM obtenidas del efluente peritoneal de los pacientes tratados con DP. Cincuenta y dos pacientes estables en DP han sido estudiados. Las CM fueron obtenidas a partir del efluente peritoneal del intercambio nocturno con glucosa al 2,27 por ciento y cultivadas en frascos de cultivo de 25 cm2. Las células de 42 pacientes (80,7 por ciento) consiguieron alcanzar la subconfluencia en cultivo. En este estadio, el porcentaje de células caracterizadas como mesoteliales, tanto morfológicamente como por técnicas de inmunohistoquímica, se incrementó hasta el 95,5 por ciento. Posteriormente, las CM se subcultivaron en placas multipocillo, donde mostraron un crecimiento exponencial hasta el día 16. En nueve pacientes (17 por ciento) el número de CM liberadas al efluente peritoneal fue tan bajo que no consiguieron alcanzar el estado de subconfluencia. Otro paciente aunque liberó un número adecuado de CM fue incapaz de alcanzar la subconfluencia. En resumen, las CM liberadas al efluente peritoneal son accesibles para su estudio mediante cultivo. Con esta técnica se abre la posibilidad del seguimiento longitudinal de la biología de las CM de forma individual en los pacientes en DP (AU)

Circulating cancer cells in peripheral blood. A case report.

BACKGROUND: Carcinocythemia, the presence of circulating cancer cells in peripheral blood, is a rare complication of solid neoplasms. When the number of such cells is very high, they can be detected during routine laboratory tests. They are associated with a dismal prognosis. CASE REPORT: Carcinocythemia occurred in a patient with disseminated breast cancer. Eighteen cases were identified from a review of the literature. The most common neoplasms associated with circulating cancer cells in peripheral blood were breast adenocarcinoma, small cell lung carcinoma and rhabdomyosarcoma. All the patients had stage IV disease at the time of diagnosis, and all had involvement of the reticuloendothelial system. Patients survived for an average of a few days or weeks. CONCLUSION: Circulating cancer cells in peripheral blood are an unusual manifestation of disseminated neoplasms that occurs as a terminal event.

Icodextrin effluent leads to a greater proliferation than glucose effluent of human mesothelial cells studied ex vivo.

OBJECTIVE: To compare the effect of glucose (Glu) and icodextrin (Ico) dialysate on in vitro culture of mesothelial cells (MC) from peritoneal dialysis (PD) patients. DESIGN: Prospective, controlled comparative study on the effects of two PD solutions. SETTING: A tertiary-care public university hospital. PATIENTS: Sixteen PD patients regularly using Glu dialysate were asked to collect an 8-hour dwell peritoneal effluent on 2 different days, with an interval shorter than 7 days. In the first collection, 2.27% Glu solution and in the last, 7.5% Ico solution was infused. Human MC were isolated from the nocturnal peritoneal effluent bags and grown ex vivo. MAIN OUTCOME MEASURES: Mesothelial cell proliferative capacity ex vivo. RESULTS: Mesothelial cells were present in all patient dialysates except that of a single patient's Glu dialysate. The number of MC drained was similar with both solutions. After the initial culture reached confluence, MC were identified in 14 and 12 patients receiving Ico and Glu, respectively. However, in 1 patient using Ico and in 2 using Glu, the MC count at this stage was so low that further subculture could not be performed. Cells from Ico-derived solutions exhibited a higher degree of proliferation than cells from Glu-derived solutions. The morphology of MC was also different. Cells from drained effluent were typical in 11 patients using Glu solution in contrast with 14 patients using Ico. At confluence, the percentages of typical appearance were 50% and 92.9% (p < 0.05) in Glu and Ico respectively. CONCLUSIONS: Mesothelial cells taken from icodextrin effluent show a greater proliferation ex vivo than those taken from glucose effluent.

Ex vivo proliferation of mesothelial cells directly obtained from peritoneal effluent: its relationship with peritoneal antecedents and functional parameters.

The peritoneal membrane requires anatomico-functional integrity to guarantee long-term stability for peritoneal dialysis (PD). Since mesothelial cells (MC) are active cells and the first part of the membrane to contact the dialysate, they are important in maintaining this stability. Mesothelial cells released daily into peritoneal effluent are able to grow in culture. This growth capacity may be related to some of the anatomicofunctional characteristics of each peritoneum. Our aim was to culture mesothelial cells taken from peritoneal effluents drained by 32 PD-stable patients, and relate this growth capacity to individual peritoneal data. Cells were taken from a residual fluid after sedimentation, washed twice with phosphate-buffered saline (PBS), and seeded into 25-cm2 tissue-culture flasks. These flasks were incubated in a humidified 5%-CO2 atmosphere. After MC confluence, cells were detached by trypsinization, passaged into 24-well plates, and finally counted. Cells were identified by morphology and immuno-histochemical characteristics. Cells from 28 out of 32 patients showed an appropriate growth in culture. Mesothelial cell confluence was reached in a mean of 18.2 +/- 8 days. After 7 days of seeding in plate wells, the cell growth showed a significant and progressive increase until day 16. Mesothelial cell growth rate was inversely related to PD duration. Neither peritonitis incidence nor other demographic characteristic were related to MC growth. Creatinine and urea mass transfer coefficients (MTC), but not ultrafiltration (UF) capacity, were significantly related to MC growth rate. In conclusion, the growth in culture of MC taken directly from PD bags is certainly possible. This growth is influenced by some of the intrinsic peritoneal characteristics derived from the peritoneal dialysis process. This tool could be useful in evaluating individual peritoneal conditions and, probably, as a method for peritoneal viability follow-up, although further research is required.

Immunomodulation of peritoneal macrophages by granulocyte-macrophage colony-stimulating factor in humans.

Colony-stimulating factors are growth factors which induce differentiation of the hematopoietic stem cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates proliferation and improves functions of neutrophils and monocyte/macrophages. A macrophage submesothelial stratum has been suggested to constitute the first line of peritoneal defense. We have tested whether intraperitoneally administered GM-CSF could increase the number and activation of peritoneal macrophages in peritoneal dialysis patients. Eight stable patients injected 17 micrograms of GM-CSF in each of their four daily CAPD bags over three days. The clinical status, the peritoneal effluent and peripheral blood cell count, membrane receptor expression, phagocytosis activity and cytokine levels were monitored at days 0, 1, 3, 10 and 28. GM-CSF administration caused a large increase in peritoneal macrophage number (89-fold mean increase after 72 hr), returning to baseline seven days after withdrawal. GM-CSF triggered an increase in the expression of CD11b/CD18 (CR3) and its counterreceptor CD54, indicating the cellular progression into a more activated state. Both the number of phagocytic cells (55 +/- 15% to 83 +/- 10%, P < 0.05) and the phagocytic index (137 +/- 29 to 255 +/- 61, P < 0.01) were also augmented. Peritoneal effluent cytokine-chemokine levels demonstrated an increase in IL-6 and MCP-1 levels while TNF-alpha, IL-1, IL-8, MIP-1 alpha and RANTES were not significantly altered. GM-CSF administration did not affect the peritoneal transport of water or solutes. Minor side-effects were registered in two patients. In conclusion, intraperitoneal GM-CSF causes a marked and transient recruitment of primed macrophages into the peritoneum without inducing inflammatory parameters. GM-CSF should improve the peritoneal defensive capacity through potentiation of the effector functions of resident and newly-recruited macrophages.

Circulating burst-forming unit-erythroid (BFU-E) colonies as an early marker to measure the recombinant human erythropoietin response in peritoneal dialysis patients.

To evaluate circulating burst-forming unit-erythroid (BFU-E) cells after erythropoietin (EPO) treatment, 7 female continuous ambulatory peritoneal dialysis patients were studied [baseline hemoglobin (HB): 7-10.5 g/dL)]. EPO (4000 U subcutaneously) was administered twice weekly for two months. The control group consisted of 7 healthy women with similar demographic characteristics. Mononuclear cells (MC) (5 x 10(5)) were added to 1 mL of culture medium, supplemented with 30% fetal bovine serum, 2 U/mL of EPO, 1% bovine serum albumin, and 0.3% agar. To enumerate colonies derived from each circulating BFU-E, plates were examined by inverse light microscopy, identifying BFU-E as large aggregates with more than 100 hemoglobinized cells (HC). A remarkable individual response was observed, with Hb values ranging 8.4-13.6 g/dL at week 4 and 8.8-16.5 g/dL at week 8. Baseline levels of BFU-E in patients ranged 0-100/5 x 10(5) MC (0-358 mL of whole blood), and in controls they ranged 6-24/5 x 10(5) MC (29.6-101.3/mL) (NS). Hemoglobin levels after four and eight weeks of EPO showed a significant relationship with circulating BFU-E at baseline (r = -0.873, r = -0.928, respectively). The increment in Hb after eight weeks showed a significant relationship with baseline BFU-E (r = 0.812). Hemoglobin levels at the fourth to eighth week, and the increment in Hb after eight weeks, showed a significant, direct relationship with the increments in circulating BFU-E registered after one to two weeks of treatment. We conclude that, after the first week of EPO treatment, its effect on hemoglobin may be predieted by the increment in circulating BFU-E. Thus; a new tool to measure the earliest EPO effects has become available.

Transformed mesothelial cells in patients on CAPD for medium- to long-term periods.

OBJECTIVE: To describe the characteristics of abnormal cells present in the peritoneal effluent of 4 continuous ambulatory peritoneal dialysis (CAPD) patients; the cells were accidentally detected in a longitudinal study of cell populations in 83 patients. DESIGN: Descriptive study. PARTICIPANTS: Four stable CAPD patients (2 male, 2 female). INTERVENTIONS: Peritoneal cells were collected from nocturnal peritoneal effluent (NPE) by centrifugation. MEASUREMENTS: Light microscopy, ultrastructural, cytochemical, and immunohistochemical characteristics were studied. RESULTS: The abnormal cells were characterized by a flat appearance, large size (diameter 100 microns)--six to ten times larger than a normal macrophage, a broad acidophilic cytoplasm with rare granulations, and a low nucleus/cytoplasm ratio. The nucleus was pyknotic, with dense chromatin and sometimes appeared fragmented. Its number presented a considerable variability between the patients and was much higher in the 2 females. This number remained stable in each patient over time. These cells were negative for beta-glucuronidase and positive for PAS stain with variable intensity. A very low number of flat cells were positive for vimentin with weak intensity, whereas cytokeratin and epithelial membrane antigen (EMA) were positive in a higher number of cells with medium to strong intensity. Ultrastructural studies showed numerous short surface microvilli, cytoplasm well-developed with intracytoplasmic lumina and abundant, dispersed intermediate filaments, scattered mitochondria, and stacks of rough endoplasmic reticulum were observed. Dispersed secretory vacuoles and isolated lipid vacuoles were present. CONCLUSION: All these features imply that they are mesothelial in origin and are suggestive of a change known as peritoneal squamous metaplasia. To date, the clinical follow-up of our patients has shown a benign outcome; further studies are necessary to elucidate the significance of this peritoneal squamous metaplasia in CAPD patients.

Cell populations present in the nocturnal peritoneal effluent of patients on continuous ambulatory peritoneal dialysis and their relationship with peritoneal function and incidence of peritonitis.

OBJECTIVE: To study the relationship between peritoneal effluent cells and infection rate and to relate this population with functional characteristics. DESIGN: Prospective, longitudinal, and comparative study. SETTING: Outpatient continuous ambulatory peritoneal dialysis (CAPD) unit of a university medical center. PARTICIPANTS: Seventy-one uninfected patients, treated for 0-156 months on CAPD, in stable condition were studied (33 female, 38 male). INTERVENTIONS: Nocturnal peritoneal effluent (NPE) was drained with EDTA (2.5 mmol/L) at 37 degrees C and centrifuged at 2500 rpm for 9 minutes. MEASUREMENTS: Accumulated peritoneal inflammation days/year and ultrafiltration/diffusion (mass transfer coefficients (MTCs) for small molecules) capacities were recorded. Cellular count (cells/night) was performed using a Neubauer chamber. Macrophage function was assessed by cytochemical (lysosomal enzyme content: ANAE, beta-glucuronidase, acid phosphatase) and immunohistochemical procedures (expression of membrane antigens, CD4, 11b, 11c, 14, 16, 25, 35, and 71). RESULTS: The macrophage is the most frequently appearing cell in the NPE. Cell count decreases over time on CAPD (from 20 x 10(6) to 5 x 10(6) after the first year). Intrapatient variability was low, but interpatient differences were marked. Mesothelial cell count remained stable over time (0.25-0.5 x 10(6)). Four of our patients showed a "transforming" change in these cells. Previous incidence of peritonitis and values of functional measurements did not correlate with cell count or expressions of macrophage function (lysosome enzyme content and percentage of cells expressing different membrane antigens). CONCLUSION: There is difficulty interpreting the results on peritoneal effluent cells and their relationship with the incidence of peritonitis and functional characteristics of the peritoneum. No definite conclusions can be drawn other than the great interpatient and intrapatient variability. The presence of abnormal peritoneal cells with undetermined origin and function suggests the need for periodic studies of peritoneal effluent cells on long-term CAPD patients.

Presence of different mitogenic activities in the nocturnal peritoneal effluent of patients treated with CAPD.

Peritoneal cells in continuous ambulatory peritoneal dialysis (CAPD) patients are continually being regenerated. We previously reported that nocturnal peritoneal effluent is mitogenic on human and mouse fibroblasts in culture, especially when a comitogen is present. The nature, origin, and role of this mitogenic activity remain undetermined. The data resulting from the addition of different comitogen to dialysate suggest that the peritoneal effluent contains different growth factors with molecular weight greater than 10,000 dalton. Also, the presence of a growth inhibitor is plausible. In conclusion, different growth-promoting and inhibiting activities are present in the peritoneal effluent, suggesting a complex cellular relationship as a result of peritoneal dialysis with unknown consequences.

[Evaluation of the Coulter STKS hematologic autoanalyzer]. / Valoración del autoanalizador hematológico Coulter STKS.

PURPOSE: To express the results of an evaluation of the Coulter STKS blood cell counter. MATERIAL AND METHODS: The following data were collected: inaccuracy, carry-over, linearity, comparison with other systems, suspect morphology alarms, stability of calibration, time-stability of samples and working velocity. The protocol recommended by the ICSH was followed for the first four of the above. RESULTS: Good results were achieved for cell counts and derived parameters. The leucocyte differential count showed fair results for neutrophils, lymphocytes, monocytes and eosinophils; false basophilia was usually associated with the presence of abnormal lymphocytes. No differences were found between adult and paediatric samples. The usefulness of the suspect morphology alarms was also evaluated; they were found fairly sensitive for the presence of stabs, large amounts of atypical lymphocytes and blast cells, and moderately sensitive for erythroblasts and platelet clumps. The predictive value of alarms was found high in any type of pathology, but scarcely valuable when considering the pathology indicated by the alarm, such predictive value being noticeably increased upon associated alarms. CONCLUSION: The Coulter STKS cell counter appears as adequate for the haematology laboratory running daily high number of samples with high pathologic rates.

[The association of myasthenia gravis and hairy-cell leukemia]. / Asociación de miastenia grave y tricoleucemia.

Myasthenia gravis (MG) and tricholeucemia (TL) produce an important immune alteration favoring the appearance of neoplastic and autoimmune disorders. The case of a 53 years old patient who developed type B TL 5 years after the diagnosis of MG is reported. Upon revision of the literature, 37 cases of association of MG and malignant hemopathies, fundamentally lymphoproliferative were found. In general, MG precedes the appearance of the neoplastic process. The autoimmunity and immunodeficiency characteristic of MG probably constitute the pathogenic mechanism leading to the appearance of the neoplastic process.

[Evaluation of the Sysmex NE-8000 hematologic autoanalyzer]. / Evaluación del autoanalizador hematológico Sysmex NE-8000.

The results of an evaluation of the Sysmex NE-8000 cell counter are reported. Good results were attained for all values of red and white cell counts. Platelet values were good as well, although samples with thrombocytosis were overstated to some extent. Fair results were found for the white-cell differential count concerning neutrophils, eosinophils and lymphocytes, but false monocytosis were frequently seen in paediatric blood samples, whereas some monocytosis were undetected. The alarms for suspected pathology had only moderate sensitivity when used as the only criterion. The predictive value of such alarms was low, except for the "platelet clumps?" sign.

Transitory T-lymphoblastic leukemoid reaction in a neonate with Down syndrome.

A transient leukemoid reaction in a neonate with Down syndrome is reported. The blastic proliferation was identified as T lymphoblastic in an early stage of maturation (prethymocytes) using morphological, cytochemical, and immunological methods. A spontaneous complete remission occurred in 8 weeks. No additional cytogenetic alterations were found, except for those concerning chromosome 21. Other cases reported in the literature reveal that cytogenetic studies may be useful to distinguish these transient leukemic reactions from true leukemias in newborns with Down syndrome. The in vitro growth pattern of peripheral blood and bone marrow may also be useful for this purpose.

[Evaluation of a totally automated alternative system for determining the rate of erythrocyte sedimentation]. / Valoración de un sistema alternativo totalmente automatizado para la determinación de la velocidad de sedimentación globular.

The Ves-MaticR system (VS) for evaluating erythrocyte sedimentation rate (ESR) is compared to Westergren method. A good correlation between both methods is obtained in spite of small differences observed; the accuracy of the VS is also studied. Concerning the time variation, a small reduction of the ESR in the first seven hours is observed, being significant at 24 hours. The most important advantages of the VS are the near complete disappearance of the contamination risk, the reduction of the time employed in setting up, sedimentation and reading, and the reliability.