Serum Insulin-Like Growth Factor-1 (IGF-1) Age-Specific Reference Values for Healthy Adult Population of Serbia.

Context: Insulin-like growth factor-1 (IGF-1) is main serum surrogate marker of growth hormone (GH) secretion, used in diagnostics and treatment of GH deficiency (GHD) and acromegaly. Regional, ethnic, racial or nutritional factors obscure cross-population applicability of IGF-1 reference values. Establishment of population- and assay-specific reference values requires sizable representative cohort of healthy subjects. Subjects and Methods: In representative sample of healthy adult population of Serbia (N=1200, 21-80 years, 1:1 male:female) serum IGF-1 was analyzed by Siemens Immulite 2000 assay under uniform laboratory conditions. Upper and lower limit of reference range (5th - 95th percentile) were calculated for each of the 12 quinquennial age intervals. IGF-1 distribution was normalized and standard deviation score (SDS) calculated by Logarithmic and LMS methods. Results: IGF-1 and age correlated significantly, with most prominent decline at 21-50 years, followed by a plateau up to age of 70. Gender differences were not significant overall. Plateau in age-related IGF-1 decline was less prominent in women. Correlations of IGF-1 with body mass index (BMI) or waist to hip ratio (WHR) were insignificant. Superior IGF-1 SDS transformation was achieved with LMS method, while logarithmic method was simpler to use. Conclusions: Normative age-specific serum IGF-1 reference values were established on a representative cohort of healthy adults in Serbia. Our results support recommendations against necessity for gender-specific or BMI- and WHR-specific reference ranges. Population-based data serve to generate IGF-1 SDS, which is valuable in rational application of consensus guidelines, proper longitudinal follow-up, advancement in efficacy and safety and personalization of treatment targets.

FROM DIABETES INSIPIDUS TO SELLAR XANTHOGRANULOMA - A "YELLOW BRICK ROAD" DEMANDING TEAM-WORK.

Xanthogranulomas are inflammatory lesions exceptionally rarely occurring in the sellar region. Sellar xanthogranulomas (SXG) result from secondary hemorrhage, infarction, inflammation or necrosis upon existing craniopharyngioma (CP), Rathkès cleft cyst (RCC) or pituitary adenoma (PA), or represent a stage in xanthomatous hypophysitis evolution. "Pure SXG" are independent of a preexisting lesion. A 70 year old male patient, laryngeal cancer survivor, presented with central diabetes insipidus (CDI). MRI revealed an intra-suprasellar mass of uncertain origin. Transsphenoidal surgery resulted in an efficient lesion resection with maximal pituitary sparing. Pathological report has confirmed SXG without conclusive identification of preexisting sellar lesion. Age at presentation and gender were atypical for SXG. The most frequent presenting signs of SXG were absent. Most SXG are initially misdiagnosed as CP, RCC or PA. Preoperative clinical and radiological uncertainty may impact operative planning. Differentiating from CP is crucial, due to divergent operative target goals and prognosis. Intraoperative frozen section analysis could guide surgical extensiveness. Close collaboration must include endocrinologist, neuroradiologist, neurosurgeon and pathologist. Quantity and quality of provided tissue are essential for avoiding bias in pathohistological analysis of cystic or heterogenous lesions. Awareness is needed of new pathological entities in the sellar-parasellar region. SXG should be considered in differential diagnosis of CDI-causing sellar lesions.

Etiology of Hypopituitarism in Adult Patients: The Experience of a Single Center Database in the Serbian Population.

There are only a few published studies related to the population-based etiology of hypopituitarism. New risks for developing hypopituitarism have been recognized in the last 10 years. Aim. To present data regarding the etiology of hypopituitarism collected in a tertiary center over the last decade. This is a cross-sectional database study. Patients and Methods. We included 512 patients (pts) with hypopituitarism, with a mean age of 45.9 ± 1.7 yrs (range: 18-82; male: 57.9%). Results. Nonfunctional pituitary adenomas were presented in 205 pts (40.5%), congenital causes in 74 pts (14.6%), while acromegaly and prolactinomas were presented in 37 (7.2%) and 36 (7.0%) patients, respectively. Craniopharyngiomas were detected in 30 pts (5.9%), and head trauma due to trauma brain injury-TBI and subarachnoid hemorrhage-SAH in 27 pts (5.4%). Survivors of hemorrhagic fever with renal syndrome (HFRS) and those with previous cranial irradiation were presented in the same frequency (18 pts, 3.5% each). Conclusion. The most common causes of hypopituitarism in our database are pituitary adenomas. Increased awareness of the other causes of pituitary dysfunction, such as congenital, head trauma, extrapituitary cranial irradiation, and infections, is the reason for a higher frequency of these etiologies of hypopituitarism in the presented database.

Taliglucerase alfa leads to favorable bone marrow responses in patients with type I Gaucher disease.

Taliglucerase alfa (Protalix Biotherapeutics, Israel) is a carrot-cell-expressed recombinant human beta-glucocerebrosidase recently approved in the United States for the treatment of type 1 Gaucher disease (GD). As bone disease is one of the most debilitating features of GD, quantification of bone marrow involvement is important for monitoring the response to treatment. Therefore, bone marrow fat fraction (Ff) measured by quantitative chemical shift imaging (QCSI) was included as exploratory parameter to evaluate bone marrow response in treatment naïve GD patients participating in a double-blind, randomized phase III study. Eight GD patients with intact spleens were treated with 30 or 60U/kg biweekly. Ff results were compared to outcomes in 15 untreated Dutch GD patients with a follow-up interval of 1year. Five taliglucerase alfa treated patients had a Ff below the threshold that relates to complication risk (<0.23) at baseline (median (n=8) 0.19, range 0.11-0.35). Ff significantly increased compared to baseline (p=0.012) and compared to untreated patients (p=0.005), already after 1year of follow-up with further improvement up to 36months. In four patients with the lowest Ff, the higher dose resulted in increases above 0.23 within 1year. All patients had sustained improvements in all other parameters. There was no influence of antibodies on response parameters. Treatment with taliglucerase alfa results in significant increases in lumbar spine fat fractions, which indicates clearance of Gaucher cells from the bone marrow.

Insulin resistance in non-obese women with polycystic ovary syndrome: relation to byproducts of oxidative stress.

To get more insight into molecular mechanisms underlying oxidative stress and its link with insulin resistance, oxidative stress parameters, as well as, antioxidant enzyme activities were studied in young, non-obese women with polycystic ovary syndrome (PCOS). Study was performed in 34 PCOS women and 23 age and body mass index (BMI)-matched healthy controls. Plasma nitrotyrosine and malondialdehyde (MDA), representative byproducts of protein and lipid oxidative damage, were determined by enzyme immunoassay. Antioxidant enzyme activities, superoxide dismutase (SOD) and glutathione peroxidase (GPX) were studied spectrophotometrically. Insulin resistance was calculated using homeostasis assessment model (HOMA-IR). Plasma nitrotyrosine and MDA were increased, but only nitrotyrosine was significantly higher (p < 0.05) in PCOS women compared to controls. Uric acid (surrogate marker of × antine oxidase) was also significantly elevated in PCOS (p < 0.05). Both plasma SOD and GPX activity showed no statistically significant difference between PCOS and controls. Indices of insulin resistance (insulin and HOMAIR) were significantly higher in PCOS group and positively correlated with level of MDA (r = 0.397 and r = 0.523, respectively; p < 0.05) as well as GPX activity (r = 0.531 and r = 0.358, respectively; p < 0.05). Our results indicate that insulin resistance could be responsible for the existence of subtle form of oxidative stress in young, nonobese PCOS women. Hence, presence of insulin resistance, hyperinsulinemia and oxidative damage are likely to accelerate slow development of cardiovascular disease in PCOS.

Collection strategies and cryopreservation of umbilical cord blood.

The aim of this study was to compare (a) two different umbilical cord blood (UCB) collection methods while the placenta is still in the uterus (in utero), and (b) to evaluate the efficacy of four cryopreservation protocols based on UCB haematopoiestic stem cell (HSC) recovery. We analysed UCB samples collected with our original collection system designed for active Syringe/Flush/Syringe method or by standard in utero method. For comparing different cryopreservation procedures, dimethyl sulphoxide (DMSO) at final concentration of 5 and 10% was used and combined with our own controlled-rate or uncontrolled-rate cryopreservation. A total of 99 samples were collected. A significantly higher UCB volume, total nucleated cell and mononuclear cell were seen following the first collection strategy (n= 49; mean +/- SD, 103 +/- 35.4 mL; 12.34 +/- 5.27 x 10(8); 595 +/- 3.47 x 10(6)) vs. the second strategy (n= 50; 86 +/- 29.3 mL; 9.87 +/- 4.47; 424 +/- 2.82 x 10(6)) respectively (P < 0.01). The discard rate was 14% for the first and 36% for the second collection strategy (P < 0.01). It was shown that the most efficient procedure was the controlled-rate protocol combined with lower (5%) DMSO concentration. Using active Syringe/Flush/Syringe method, we collected UCB with greater volumes and with lower discard rate compared to the standard by gravity technique. The data presented also showed much better recovery of UCB cells when controlled-rate freezing procedure and 5% DMSO were combined.

The effect of interleukin-17 on hematopoietic cells and cytokine release in mouse spleen.

To evaluate whether the response of hematopoietic cells to interleukin-17 (IL-17) depends on the tissue microenvironment in which hematopoiesis occurs, the influence of recombinant mouse IL-17 on spleen hematopoietic cells and cytokine release was assessed in normal mice in vitro and in vivo. In vitro, IL-17 did not significantly affect the growth of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) derived colonies. A single injection of IL-17 in vivo exhibited stimulatory effects on hematopoietic cells from both granulocytic and erythroid lineages. The increased number of metamyelocytes 48 h after treatment imply to the IL-17-induced stimulation of granulopoiesis. The number of BFU-E was increased at 24 h, while the number of CFU-E increased 6 h and 24 h after treatment. Since the same treatment in the bone marrow decreased the number of CFU-E, it may be concluded that the local microenvironment plays an important role in IL-17-mediated effects on CFU-E. IL-17 increased the release of IL-6 both in vitro and in vivo, but showed tendency to suppress the constitutive secretion of IL-10 by spleen cells. Our results suggest the complexity of target cell response and interplay of secondary induced cytokines by IL-17 in different hematopoietic organs.

[Endoscopic ultrasonography (EUS) in preoperative localization of neuroendocrine tumors (NET) of the pancreas].

BACKGROUND: Preoperative localization of pancreatic neuroendocrine tumours (NET) is usually very difficult. Noninvasive, imaging tests, such as abdominal ultrasound, CT or MRI are not sensitive enough as well as selective angiography. The aim of the study was to clarify the usefulness of the EUS in preoperative localization of the pancreatic NET. METHODS: From September 1998 March 2005, EUS was performed in 1600 patients. Among them, in 10 (0.7%), this examination was carried out due to previous biochemical tests, which diagnosed the pancreatic NET. We studied the location, the size and echo-pattern of the neoplasm. The results were compared with operation and histology or EUS- FNA guided pancreatic biopsy in 9/10 patients. All EUS examinations were performed using Olympus GIF-130 videoecho-endoscope with 7,5 /12MHz switchable radial probe. RESULTS: EUS correctly localized the pancreatic NET in 7/8 cases, (sensitivity:87.5%). In 2 patients, EUS accurately exclouded pancreatic NET. There were no false positive findings (specificity 100%). Six tumours were benign (75%), and two were malign (25%). We localized 6 insulinomas and single pancreatic carcinoid tumour. The median tumour size detected by EUS was 21mm. CONCLUSION: EUS is highly accurate in preoperative localization of the pancreatic NET-s and We confirmed it in our study. EUS presents the method of choice for preoperative localization of the pancreatic NET.

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice.

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.

Gonadotropin response to clomiphene and plasma leptin levels in weight recovered but amenorrhoeic patients with anorexia nervosa.

Anorexia nervosa (AN) is a state of leptin and gonadotropin deficiency. Leptin levels are decreased in normal weight women with hypothalamic amenorrhea and leptin may be a sensitive marker of overall nutritional status. The aim of the study is to provide additional information on plasma leptin levels and on gonadotropin responses after clomiphene testing in patients with AN who recovered weight but were still amenorrheic. We evaluated 17 patients with AN, female age 20+/-1.2 yr who reached goal weight [body mass index (BMI) 14.9+/-0.5 to 19.3+/-0.4 kg/m2]. At diagnosis serum leptin levels were 2.2+/-0.1 microg/l while after behavioural therapy and hypercaloric diet for 6-12 months serum leptin levels rose to 6.4+/-1.4 microg/l significantly lower compared with those in the control (no.=10, age 28+/-6.2 yr, BMI 21.1+/-0.3 kg/m2, leptin 9.3+/-0.7 pg/l; p<0.05). None of the patients resumed spontaneous menstrual cycles after weight gain. They were tested with a 10-day administration of clomiphene citrate. All had a significant rise in LH secretion (from 1.7+/-0.3 IU/l to 8.3+/-0.9 IU/l, p<0.01) and serum estradiol levels (from 19.0+/-5.4 to 937.7+/-241.2 pg/ml, p<0.03). Nine out of 17 patients menstruated after clomiphene. Serum leptin levels were not different in those who menstruated from those who did not (6.4+/-1.4 to 6.8+/-1.4 microg/l, p>0.05). Body compositon was studied in 12 additional carefully matched patients with AN who recovered weight. Six of them resumed spontaneous menstrual cycles. Neither BMI, body fat, nor leptin appeared as significant determinants of menstrual status. In conclusion, relative hypoleptinemia persists, independent of fat mass, in weight recovered patients with AN. A normal response to clomiphene in weight-recovered yet still amenorrhoeic patients with AN, offers reassurance that the axis is intact and that the problem lies in the hypothalamus. It is reasonable to believe that nutritional disturbances, fat intake and persisting psychological factors still affect plasma leptin levels and reproductive functions in weight-recovered patients with amenorrhea.

Case report of hypopituitarism with suspected syndrome of inappropriate VP secretion (SIADH) due to a large aneurysm of the internal carotid in the sellar region.

Hypopituitarism and hyponatremia, especially when severe, are infrequent findings particularly when the cause of hypopituitarism at presentation is unknown and untreated. Interestingly, hyponatremia is usually seen in elderly patients with hypopituitarism due to various causes. We present a case with unrecognized and untreated hypopituitarism due to a large aneurysm of the internal carotid artery in the sellar region causing the syndrome of inappropriate secretion of antidiuretic hormone (SIADH).

Hematopoiesis during acute Toxoplasma gondii infection in mice.

We studied hematopoiesis in bone marrow and blood of CBA mice following infection with Toxoplasma gondii. Our data showed that acute infection with the virulent RH strain was associated with leucopenia, thrombocytopenia and bone marrow hypoplasia while, in spite of the infection-induced damage of the granulocyte cell lineage, in bone marrow stimulated production of granulocytes was revealed. In peripheral blood, T. gondii infection caused a significant decrease in the total number of white blood cells, reticulocytes and platelets. However, the relative proportion of granulocytes and lymphocytes was changed in favor of granulocytes, as compared to pre-infection levels. The functional activity of granulocytes was also increased. The bone marrow alterations were characterized by a decrease in the total number of nucleated cells due to the reduced numbers in all cell compartments of erythroid and megakaryocytic lineage, as well as in the number of mature granulocytes and lymphocytes. In contrast, femoral granulocytic proliferative compartments, colony forming unite granulocyte-macrophage (CFU-GM) and morphologically recognizable proliferative granulocytes (PG), exhibited stimulated granulopoiesis, while the number of mature monocytes was close to the control value. In summary, we have shown that acute T. gondii infection results in profound alterations of the hematopoietic system that markedly contribute to the clinical onset of the disease and the, ultimately lethal, outcome.

Effect of IL-17 on in vitro hematopoietic progenitor cells growth and cytokine release in normal and post-irradiated murine bone marrow.

The influence of recombinant human IL-17 on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitors and the release of IL-1alpha/beta, IL-6 and erythropoietin (EPO) was estimated in the bone marrow cells obtained from normal and sublethally irradiated mice. In normal mice IL-17 increased CFU-GM and BFU-E and reduced CFU-E derived colonies numbers and augmented release of IL-6 and EPO. In irradiated mice the effects of IL-17 on hematopoietic progenitors were lineage-dependent, as well as dependent on their stage of differentiation and the time after the irradiation. IL-17 had no major effects on CFU-GM on day 1 and 3, but decreased their number on day 2, while enhanced both BFU-E and CFU-E on day 1 and 2 after irradiation, whereas on day 3 its effect on erythroid progenitors was again as observed in normal mice. After irradiation, IL-17 increased the release of IL-1alpha, IL-6 and EPO. The observed effects suggested the involvement of IL-17 in the regulation of hematopoiesis and indicated that its effects on both hematopoietic progenitors and cytokine release are dependent on the physiological/pathological status of the organism.

Loss of gender difference in serum leptin levels and its slow recovery after successful surgery for Leydig cell tumours in two virilized females.

One of the factors that predicts serum leptin levels is gender. It has been shown that sex steroid hormones, in particular testosterone, play an important role in the regulation of serum leptin levels. We had the opportunity to examine the effects of acute and chronic changes in serum testosterone levels on serum leptin concentrations in two virilized females harbouring testosterone-secreting ovarian tumours, before and after curative surgery. Chronically elevated basal testosterone levels (46 nmol/l) were associated with suppressed serum leptin levels (1.46 microg/l and 2.56 microg/l) vs. 12 age- and BMI-matched healthy subjects 9.89 +/- 0.64 microg/l. Leptin levels were determined from pooled serum samples assayed by commercial radioimmunoassay. High testosterone levels abolished the well known sexual dimorphism of serum leptin levels. Two weeks after curative resection of these tumours serum leptin levels were unaltered and started to increase progressively after one month. One patient received parenteral conjugated oestrogens while the other resumed spontaneous menstrual cycles. Three months after curative surgery obvious changes in body composition were registered (DEXA). Six months later further rise in serum leptin concentrations occurred without further changes in body composition. In conclusion, leptin levels did not change in spite of rapid changes in the steroid milieu, but in the long term increase in body fat stores, new steroid milieu and maybe other factors are important determining factors of serum leptin levels.

Effects of treatment with interleukin-1 receptor antagonist on endogenous interleukin-1 levels in normal and irradiated mice.

The in vivo effects of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) administration on endogenous IL-1 levels in the circulation and conditioned media (CM) from different immunohematopoietic organ/tissues were studied in CBA mice under steady state and postirradiation conditions. In normal mice, constitutive IL-1 levels were demonstrated in the plasma, CM of peritoneal exudate cells and full-thickness skin explants with low or undetectable levels in CM of splenic and bone marrow cell suspensions. In irradiated mice (2 Gy, X rays) on day 3 post exposure a significant increase of IL-1 levels was seen in the circulation and CM of peritoneal exudate cells, with no significantly different levels in postirradiation bone marrow, spleen and skin. After rhIL-1Ra treatment of the animals (2 x 50 microg/mouse, i.p.), significantly elevated IL-1 levels were observed in the skin and CM of peritoneal exudate cells in normal mice, whereas slightly increased levels were detected in CM of splenic cells. The rhIL-1Ra administration in irradiated mice led to decreased IL-1 concentrations in the circulation, and CM of peritoneal exudate cells and skin. The results pointed out the importance of IL-1 secretion and receptor expression in the maintenance of homeostasis in steady state, as well as during recovery after irradiation. Modulatory effects of IL-1Ra on IL-1 production were dependent on basic endogenous IL-1 concentration.

Serum leptin levels in patients with acromegaly before and after correction of hypersomatotropism by trans-sphenoidal surgery.

It has been shown that GH excess is associated with decreased leptin levels and decreased body fat mass. Reports regarding the effect of GH on serum leptin levels are inconsistent. We studied leptin secretion in 20 acromegalics before and 2 months after trans-sphenoidal surgery and in 20 gender-, age-, and body mass index (BMI)-matched control subjects. The mean 8-h leptin concentration for each subject was measured from a pool formed of samples collected hourly beginning at 2200 h until 0600 h the next morning. In a subgroup of 10 acromegalics, leptin pulsatility was assessed for the same period of time in 10-min sampling intervals. Basal GH, insulin-like growth factor-I (IGF-I), insulin, glucose, and lipids levels were measured. Area under the curve for insulin (AUCins) during oral glucose tolerance test was calculated. Control subjects and acromegalics had similar BMI, but patients with active acromegaly had significantly lower mean leptin level (mean +/- SEM; in men, 2.6+/-0.4 vs. 7.1+/-1.1 microg/L, P = 0.003; in women, 16.0+/-3.4 vs. 23.5+/-3.1 microg/L; P = 0.036). Mean 8-h leptin correlated with BMI (r = 0.57, P = 0.007, in controls; r = 0.70, P = 0.001, in patients). In stepwise regression analysis with mean 8-h leptin as a dependent variable, BMI (P<0.001) and gender (P = 0.01) in acromegalics entered the equation, whereas in control subjects gender, free fatty acids, insulin, and age accounted for 99.3% in leptin variability. After surgery, BMI did not change significantly; and glucose (P = 0.014), GH (P<0.001), and IGF-I (P<0.001) levels together with AUCins (P = 0.002) decreased, whereas mean leptin concentration rose significantly and attained normal levels (4.1+/-0.8 microg/L, P = 0.028) in acromegalic men and (23.6+/-4.7 microg/L, P = 0.003) in acromegalic women. Correlation between leptin level and BMI was preserved after surgery (r = 0.62, P = 0.005). In stepwise regression analysis, free fatty acids (P = 0.04) contributed to 26.8% of the variance in corrected-leptin (for BMI and gender). Leptin concentration peak height and interpeak nadir level rose significantly (P = 0.033 and P = 0.037) after surgery by Cluster analysis, without significant changes in leptin pulse frequency and incremental peak amplitude. Nocturnal rise of leptin (mathematically described by a cubic curve) was characterized by an acrophase just after midnight, before and after surgery. The amplitude and the average leptin concentration of the cubic fit increased significantly after surgery (P = 0.028 and P< 0.001). In conclusion in acromegalic patients: 1) leptin secretion maintains the pulsatility and nocturnal rise; 2) the gender-based leptin differences are preserved; 3) GH-IGF-I normalization leads to a rise in leptin that is not related to changes in BMI; and 4) the possible role of rise in leptin levels when assessing clinical and metabolic outcome of therapy in acromegalic patients deserves additional studies.

Donor leukocyte infusion--the effect of mutual reactivity of donor's and recipient's peripheral blood mononuclear cells on hematopoietic progenitor cells growth.

Donor leukocyte infusions are an effective therapy for patients who relapse with leukemia after bone marrow transplantation. We report the case of 14-year-old boy who relapsed 34 months after sibling donor bone marrow transplant for Philadelphia-positive chronic myeloid leukemia. Subsequently, he received three infusions of donor mononuclear cells (DMNC) harvested in steady state hematopoiesis and one G-CSF mobilized-peripheral blood mononuclear cells (PBMC) infusion. Simultaneously, test named as--"Test of Mixed Progenitors" (TMP) was performed for the assessment whether the outcome of donor leukocyte infusion treatment could be predicted. Prior to DMNC infusions, the CFU-GM and BFU-E colony assays were performed for donor's and recipient's PBMC individually, as well as for the mixture of these cells at 1:1 ratio. The cells were plated either directly in the semisolid medium or after 24 h preincubation treatment. Significantly lower values for CFU-GM derived colonies were determined in TMP in comparison to the CFU-GM values obtained for the recipient's cells. The reduced number of CFU-GM was determined both in TMP performed without preincubation treatment, app. 80% and after the 24 h preincubation, app. 55%. The reduced number of BFU-E derived colonies (app. 44%) was observed only related to recipient's cells and after the preincubation treatment of the cells. The patient did not develop GVHD and currently (40 months after the first infusion). He remained well in complete hematological, cytogenetic, molecular and clinical remission, which was the most direct evidence of the GVL effect. The novel in vitro TMP test in which the specific contribution of donor's leukocytes to the growth of recipient's hematopoietic precursor cell growth was determined, correlated with the clinical outcome.

Cytochemical & ultrastructural alteration of cytoplasmic granules of rat peripheral blood neutrophils induced by chronic alcoholism & malnutrition.

The specific influence of malnutrition on the pathophysiologic changes induced by chronic alcoholism is controversial. In an attempt to determine and demarcate the effects of protein malnutrition from those produced by alcoholism and to evaluate the precise effect of alcohol per se on cytochemical and ultrastructural properties of rat polymorphonuclear neutrophil (PMN) granules, we investigated the influence of chronic protein malnutrition or chronic alcoholism alone and in combination, in rats. After a 4 month experimental period various PMN properties, such as cytochemical, morphometrical and ultrastructural, as well as neutrophil functions were studied. It was found that the degree of damage of PMNs induced either by ethanol or protein malnutrition alone was similar whereas their combination led to worsening of all markers of PMN functional ability. Ultrastructural changes of neutrophil granules including reduction, redistribution and atypical accumulation as well as appearance of autophagic vacuoles, confirmed their alteration which was emphasised by the additive pathophysiological interaction of alcoholism and chronic hypoprotein malnutrition.

The in vivo effect of liposomes on hematopoiesis.

The influence of liposome structure on hematopoiesis in vivo was assessed in relation to the different contents and origins of phospholipids that make up their membrane structures. Changes within different hematopoietic cells and serum tumor necrosis factor alpha (TNF-alpha) levels were estimated up to 14 days following intravenous administration of liposomes made of either pure egg yolk phosphatidylcholine (LEY) or a soybean phospholipid preparation (LSB) into normal CBA mice. In peripheral blood, only transient changes within white blood cells were observed. In bone marrow, a persistent decline in the number of mature granulocytes, monocytes, and lymphocytes was found. The changes within femoral granulocytic proliferative compartments in various stages of differentiation and a maturation compartment pointed out that, parallel with the depletion of the granulocyte-storage pool, stimulation of de novo production of granulocytic cells occurred. Although both types of tested liposomes induced similar cellular changes, only liposomes made of pure egg yolk phosphatidylcholine induced a transient increase in serum TNF-alpha levels.

The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating ability.

The efficiency of five different cryopreservation protocols (our original controlled-rate and noncontrolled-rate protocols) was evaluated on the basis of the recovery after thawing of very primitive pluripotent hemopoietic stem cells (MRA(CFU-GM), pluripotent progenitors (CFU-Sd12) and committed granulocyte-monocyte progenitors (CFU-GM) in mouse bone marrow. Although the nucleated cell recovery and viability determined immediately after the thawing and washing of the cells were found to be similar, whether controlled-rate or noncontrolled-rate cryopreservation protocols were used, the recovery of MRA(CFU-GM), CFU-Sd12 and CFU-GM varied depending on the type of protocol and the cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better MRA(CFU-GM), CFU-Sd12 and CFU-GM recovery from frozen samples. The most efficient was the controlled-rate protocol of cryopreservation designed to compensate for the release of fusion heat, which enabled a better survival of CFU-Sd12 and CFU-GM when combined with a lower (5%) DMSO concentration. On the contrary, a satisfactory survival rate of very primitive stem cells (MRA(CFU-GM)) was achieved only when 10% DMSO was included with a five-step protocol of cryopreservation. These results point to adequately used controlled-rate freezing as essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of very primitive stem cells, but not, however, for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in primitive MRA cells, which is not the case for less primitive progenitors.