Characterization of adenylyl cyclase stimulated by VIP in rat and mouse peritoneal macrophage membranes.

Vasoactive intestinal peptide (VIP) stimulated adenylyl cyclase activity in rat and mouse peritoneal macrophage membranes. GTP potentiated the stimulatory effect of VIP so that it was routinely included at 10 microM GTP. Other agents like GTP, Gpp(NH)p, GTP-gamma-S, sodium fluoride, and forskolin, at a concentration of 0.1 mM, increased the basal activity of enzyme by 3.1, 5.7, 4.7, 3.6, and 7.8-fold, respectively. The stimulation of adenylyl cyclase by VIP was time, temperature, and membrane concentration dependent. Half-maximal enzyme activation (ED50) was very similar in rat and mouse peritoneal macrophage membranes (1.5 +/- 0.1 nM and 1.0 +/- 0.1 nM, respectively). However, VIP showed more efficacy in mouse macrophages membranes (about 3.1-fold basal values) than that in rat macrophage membranes (about 2.5-fold basal values). The relative potency of several peptides upon stimulation of adenylyl cyclase activity showed the following potency in both species: VIP = PACAP38 = PACAP27 > helodermin > PHI > secretin. On the other hand, a M(r)-45 kDa alpha s subunit of Gs protein was demonstrated by both ADP-ribosylation and immunoblot in mouse and rat peritoneal macrophage membranes. The present results, together other previous, strongly suggest that VIP play an important role in the regulation of macrophage function.

Ontogenic development of the adenylyl cyclase enzyme and the alpha s, alpha i1 and alpha i2 G-protein regulatory subunits from rat prostate.

The expression of alpha s, alpha i1 and alpha i2 G-protein subunits measured by immunoblot increased in the rat prostate during sexual maturation, supporting their involvement in proliferation/differentiation. Northern blotting gave transcripts of 1.8 and 4 kb for alpha s, 1.4 and 4.5 kb (mainly) for alpha i1, and 2.4 kb for alpha i2 with levels suggesting a differential regulation (at transcription or post-transcription for alpha s, transcription for alpha i1, and translation for alpha i2). The stimulatory effects of forskolin, vasoactive intestinal peptide (VIP) and isoproterenol on adenylyl cyclase activity increased between 0.5-3 mo, remained constant up to 12 mo and decreased thereafter, conceivably following the expression of VIP and beta-adrenergic receptors. However, G-protein activation of adenylyl cyclase (by GTP and Gpp[NH]p) was maximal at 0.5 mo and then decreased as it occurred with toxin-catalyzed ADP-ribose incorporation to alpha subunits suggesting that other factors are also involved in the regulation of G-protein activity during rat prostatic development.

Lindane decreases forskolin-stimulated cyclic AMP accumulation but does not modify Gs in rat enterocytes.

Treatment of isolated rat enterocytes with the halogenated insecticide lindane (the gamma-isomer of hexachlorocyclohexane, HCCH) did not modify the general membrane fluidity (as estimated by a fluorescence polarization technique) nor the guanine nucleotide binding regulatory protein Gs (as studied by both ADP-ribosylation of its alpha subunit by cholera toxin and Gpp[NH]p stimulation of membrane adenylate cyclase activity). However, lindane decreased in a dose-dependent manner the effect of the diterpene forskolin on direct activation of the adenylate cyclase catalytic subunit. After 5 min of cell treatment with 0.5 mM lindane, the maximal stimulatory effect of forskolin (at 100 microM) decreased by about 50%. There was a certain degree of specificity since delta-HCCH was indeed more potent, whereas dieldrin and endrin (non-lindane related halogenated compounds) behaved as lindane, and alpha- and beta-HCCH were poorly efficient on the inhibition of forskolin stimulation of adenylate cyclase activity. A similar effect of lindane was observed on receptor-stimulated cyclic AMP accumulation by using vasoactive intestinal peptide instead of forskolin. The results on a non-receptor mediated effect of lindane on the adenylate cyclase catalytic subunit itself could be related to: (i) alterations of membrane microdomains surrounding this and other integral proteins which would result in modifications of their activities; and/or (ii) a reciprocal relation between the two main routes of signal transduction so that the activation of protein kinase C (or other Ca(2+)-dependent protein kinases) by lindane would lead to phosphorylation of the adenylate cyclase catalytic subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

[Macronutrients intake in school teenagers in Soria capital]. / Ingesta de macronutrientes en adolescentes escolarizados en Soria capital.

OBJECTIVES: To study the macronutrients intake in Soria teenagers from 10 to 19 years, as well as their body mass index (BMI). METHODS: A seven-day diet questionnaire filled in by an accidental sample of teenagers (54 boys and 56 girls) from public schools in the capital. Working out the average daily intake of energy, carbohydrates, lipids and proteins by the software of "Alimentación y Salud" which also gives values of individual recommended dietary allowances (RDAs) related to each individual's particular characteristics. Use of Student's t-test to compare the average values of the estimated intakes of different nutrients and their RDAs. RESULTS: In general, the intakes of energy, proteins and lipids are statistically significant over the RDAs, while the carbohydrates intake is under the recommendations. With reference to the type of lipids, the intake is over the RDAs for cholesterol, monounsaturated fatty acids, and saturated fatty acids, but not for polyunsaturated fatty acids. Among girls from 13 years of age more than 12% have a higher BMI than 26 kg/m2, but between 10 and 12 years of age more than 20% of the students have this parameter under 16 kg/m2. CONCLUSIONS: According to the results, it would be useful to implement some nutritional intervention among the adolescents in Soria capital to promote a healthy feeding in order to avoid possible disorders (obesity, anorexia, etc.).

Complexity and diversity of F8 genetic variations in the 1000 genomes.

BACKGROUND: Hemophilia A (HA) is an X-linked bleeding disorder caused by deleterious mutations in the coagulation factor VIII gene (F8). To date, F8 mutations have been documented predominantly in European subjects and in American subjects of European descent. Information on F8 variants in individuals of more diverse ethnic backgrounds is limited. OBJECTIVES: To discover novel and rare F8 variants, and to characterize F8 variants in diverse population backgrounds. PATIENTS/METHODS: We analyzed 2535 subjects, including 26 different ethnicities, whose data were available from the 1000 Genomes Project (1000G) phase 3 dataset, for F8 variants and their potential functional impact. RESULTS: We identified 3030 single nucleotide variants, 31 short deletions and insertions (Indels) and a large, 497 kb, deletion. Among all variants, 86.4% were rare variants and 55.6% were novel. Eighteen variants previously associated with HA were found in our study. Most of these 'HA variants' were ethnic-specific with low allele frequency; however, one variant (p.M2257V) was present in 27% of African subjects. The p.E132D, p.T281A, p.A303V and p.D422H 'HA variants' were identified only in males. Twelve novel missense variants were predicted to be deleterious. The large deletion was discovered in eight female subjects without affecting F8 transcription and the transcription of genes on the X chromosome. CONCLUSION: Characterizing F8 in the 1000G project highlighted the complexity of F8 variants and the importance of interrogating genetic variants on multiple ethnic backgrounds for associations with bleeding and thrombosis. The haplotype analysis and the orientation of duplicons that flank the large deletion suggested that the deletion was recurrent and originated by homologous recombination.

Characterization of vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptors in human benign hyperplastic prostate.

Vasoactive intestinal peptide (VIP) is an important member of the group of neuropeptides that appears to be involved in the regulation of prostatic growth and function. Here we studied VIP receptors in membranes from human benign hyperplastic prostate. Accordingly to observations in rat prostatic membranes, [125I]VIP binding to human prostatic membranes suggested two classes of binding sites with high Kd = 0.22 nM) and low (Kd = 37.7 nM) affinities. VIP bound in human and rat membrane preparations to a common VIP/pituitary adenylate cyclase-activating peptide (PACAP) receptor, as VIP, PACAP-27, and PACAP-38 were equipotent for competition of [125I]VIP binding. A PACAP-preferring receptor appears to be expressed in human prostate, since [125I]PACAP binding was displaced with more potency by PACAP than by VIP, and a messenger RNA corresponding to type I PACAP receptor was found. Cross-linking experiments suggested a VIP receptor of about 71 kDa in human and 52 kDa in rat prostates. The binding of [125I]VIP to membranes and the labeling of the bands observed after electrophoresis were competitively inhibited by GTP, suggesting the coupling of VIP receptors to a G protein. Moreover, after solubilization and cross-linking, we observed a 120-kDa band that corresponded to the VIP receptor-alpha s association. VIP stimulated adenylyl cyclase activity in a dose-dependent manner, but the potency and/or the efficacy of VIP were lower in all human preparations studied than in rat prostatic membranes. In conclusion, this study clearly demonstrates the expression of VIP/PACAP common receptors associated with alpha s protein in human prostate and suggests that these neuropeptides could play an important and complex role in the physiology and pathophysiology of this human gland.

Cloning and functional characterization of the human VIP1/PACAP receptor promoter.

The 5'-flanking region (1.5 kb) of the gene coding for the human VIP1/PACAP receptor was isolated, sequenced, and characterized. Transient expression of constructs containing sequentially deleted 5'-flanking sequences of the VIP1/PACAP receptor fused to a luciferase reporter gene showed that this sequence was active as a promoter in the intestinal cancer cell line, HT-29, expressing endogenous VIP1/PACAP receptor. The shortest DNA fragment with significant promoter activity encompassed the region from -205 to +76 bp. Deletion of a CCAAT-box sequence in the construction corresponding to -173 to +76 bp dramatically reduced the promoter activity. The promoter -205 to +76 bp has a housekeeping gene structure without TATA-box. It contains GC-rich regions characterized by potential Sp1 and AP2 sites and some potential regulatory elements, such as CRE and ATF, and a CCAAT-box sequence (-182 to -178) crucial for gene transcription.

Immunohistochemical localization and distribution of VIP/PACAP receptors in human lung.

VIP and PACAP are distributed in nerve fibers throughout the respiratory tract acting as potent bronchodilators and secretory agents. By using RT-PCR and immunoblotting techniques, we have previously shown the expression of common VIP/PACAP (VPAC(1) and VPAC(2)) and specific PACAP (PAC(1)) receptors in human lung. Here we extend our aims to investigate by immunohistochemistry their localization and distribution at this level. A clear immunopositive reaction was obtained in human lung sections by using either anti-VPAC(1) or -VPAC(2) receptor antibodies but not with anti-PAC(1) receptor antibody. However, PAC(1) receptor (and VPAC(1) and VPAC(2) receptors) could be identified in lung membranes by immunoblotting which supports that the PAC(1) receptor is expressed at a low density. Both VPAC(1) and VPAC(2) receptors showed similar immunohistochemical patterns appearing in smooth muscle cells in the wall of blood vessels and in white blood cells (mainly in areas with inflammatory responses). The results agree with previous evidence on the importance of both peptides in the immune system and support their anti-inflammatory and protective roles in lung.

Multiple regulation of adenylyl cyclase activity by G-protein coupled receptors in human foetal lung fibroblasts.

The pharmacological profile of adenylyl cyclase activity was analysed in WI-38 human foetal lung fibroblasts. Among various agents that act through G-protein coupled receptors, only the beta-adrenergic agonist isoproterenol stimulated and the tetradecapeptide somatostatin (SRIF, sst) inhibited the enzyme activity. The use of the reverse transcription-polymerase chain reaction (RT-PCR) methodology with appropriate cDNAs allowed us to identify the expression of four subtypes of SRIF transmembrane receptors (sst1-4 but not sst5 receptors) in this cell line. By RT-PCR and immunochemistry techniques, we also demonstrated the expression of stimulatory (alpha(s)) and inhibitory (alpha(i1), alpha(i2) and alpha(i3)) G-protein subunits. The known role of the adenylyl cyclase system in cell proliferation and differentiation mechanisms together with the present analysis of the corresponding regulatory network in fibroblasts of human foetal lung add knowledge on the cell line WI-38 that is widely used as a model system in studying cell growth. The importance of this cell class in normal and abnormal lung function and development reinforces the significance of these results.

Effect of bilateral adrenalectomy on VIP receptor/effector system in rat intestinal epithelial cells.

The number of vasoactive intestinal peptide (VIP) receptors and the efficiency of VIP in the stimulation of cyclic AMP accumulation in rat jejunal epithelial cells increased after bilateral adrenalectomy. However, this condition increased neither receptor affinity nor VIP potency. In addition, jejunal VIP levels followed a parallel increase. These changes reversed to control conditions after glucocorticoid replacement with dexamethasone indicating that adrenalectomy modifies the intestinal VIP receptor/effector system and suggest a relationship between corticosteroids and VIP in the functions of intestinal epithelium.

Effects of lindane on the accumulation of cyclic AMP, induced by vasoactive intestinal peptide, in isolated rat prostatic epithelial cells.

Lindane pretreatment of isolated rat prostatic epithelial cells resulted in a time- and dose-dependent impairment of the stimulation of cyclic AMP levels by vasoactive intestinal peptide (VIP); the optimal conditions for producing this impairment were found to be 5 min of cell exposure to 0.2 mm-lindane at 25 degrees C. The inhibitory effect of the insecticide was related to a decrease in VIP efficiency since the maximal cyclic AMP response (at 100 nm VIP) was about 50% of that in control cells. VIP potency was unaffected since the half-maximal cyclic AMP response was elicited at about 3 nm VIP in both the control and lindane-pretreated cells. The results indicate that lindane interferes with the cyclic AMP system in prostatic epithelium, and this could be a consequence of some interaction in the prostate gland between the two main systems for signal transduction (i.e. adenylate cyclase and phosphatidylinositol/Ca(2+)/protein kinase C).

Impairment of adenylate cyclase activity and G-proteins in human uterine leiomyoma.

The mechanisms responsible for the growth of uterine leiomyoma (a frequent cause of infertility in women) are largely unknown. Some data supports that cAMP plays a role in the growth of uterine cells but there are no reports on the status of the cAMP producing system in this human benign neoplasia. In this study, biopsies from leiomyoma and the adjacent myometrium were taken from menstruating women subjected to total hysterectomy for leiomyoma. Adenylate cyclase activity was determined by a protein-binding method, and the expression of alpha(s), alphai1/2, alphai3 and alphai0) G-protein subunits was analysed by immunoblot. The leiomyoma samples exhibited a decreased expression of as and ai1/2 with respect to the adjacent myometrial tissue. No differences were observed in alphai3 and alphaio protein expression. The basal adenylate cyclase activity as well as the efficacy (as assessed by the maximal stimulation levels) of either forskolin or, to a lesser extent, Gpp[NH]p on stimulation the enzyme activity was significantly lower in leiomyoma than in myometrium, whereas the potency (as assessed by the ED50 values) of these two agents did not vary. Present data indicate that the human leiomyoma is associated with low levels of cAMP. It is conceivable that the loss of sensitivity of adenylate cyclase to endogenous regulatory molecules could be related to the pathogenesis of human leiomyomas given that cAMP inhibits the MAP-kinase cascade in uterine tissues.

ACE: a peer education and counseling program meets the needs of incarcerated women with HIV/AIDS issues.

In this article, female prisoners who are peer educators and counselors in an HIV/AIDS program at Bedford Hills Correctional Facility, New York State's only maximum security prison for women, describe the positive role of a peer support program. Using examples from their own experiences, the women discuss the strengths of the AIDS Counseling and Education Program (ACE) in meeting the medical and psychosocial needs of the prison population concerning HIV/AIDS. The role of nurses in a correctional setting is discussed throughout the article and a final section discusses how nurses working together with peer health workers can create an effective team to meet the challenges of the AIDS epidemic within a correctional setting.

[Familial benign partial epilepsy of early infancy]. / Epilepsia parcial benigna familiar de la infancia temprana.

INTRODUCTION AND CLINICAL CASES: We present two patients who at the ages of 5 and 17 months respectively presented with convulsive crises with motor signs, of partial onset and secondary generalization, which eventually became normal. Both patients had a family history of first degree relatives with similar illnesses and are at present-five years later-well and with normal development, school achievement and neurological examination findings. The clinical characteristics, normal biochemical and neuroimaging investigations and EEG characteristics suggest the diagnosis of benign partial epilepsy of early infancy. This syndrome is characterized by its appearance during the first year of life, having no known etiological factors, with partial crises occurring several times a day and with a course leading to remission. Its frequency may be greater than is thought. There is a pattern of dominant autosomal inheritance, with a gene recently found on chromosome 19. CONCLUSION: We consider that this syndrome should be included in the International Classification of Epilepsy and Epileptic Syndromes as benign familial idiopathic partial epilepsy.

Oligomers of ß-amyloid protein (Aß1-42) induce the activation of cyclooxygenase-2 in astrocytes via an interaction with interleukin-1ß, tumour necrosis factor-α, and a nuclear factor κ-B mechanism in the rat brain.

Despite growing evidence indicating the effects of cytokines, including interleukin-1beta (IL-1ß) and tumour necrosis factor-α (TNFα), and the enzyme cyclooxygenase-2 (COX-2) in Alzheimer's diseases, little is known about the signalling mechanisms that mediate its activation in response to beta-amyloid protein (Aß). The aim of this study was first to investigate whether Aß1-42 peptide induced the up-regulation of COX-2. We then examined the expression of COX-2 and cytokines, such as IL-1ß and TNFα, in reactive astrocytes. Finally, we analyzed the role of nuclear factor kappa-B (NF-κB) as a signalling pathway in early stages of Aß-toxicity. In Wistar rats anaesthetised with equitesine, a single microinjection of Aß1-42 oligomers was made in the left retrosplenial cortex. Control animals were injected with Aß42-1 peptide into the corresponding region of the cerebral cortex. By COX-2 immunoblotting, we detected two immunopositive protein bands, at 70 and 50 kDa molecular mass. In the Aß1-42-injected animals the 50 kDa fragment showed a significant increase at 3 and 14 days, as compared with that seen in control animals. The 70 kDa fragment showed a maximal increase at 14 days. In the Aß1-42-injected animals immunoblot staining of NF-κB detected an active protein band at 50 kDa molecular mass, showing a maximal increase at the 72 h time point. Confocal analysis revealed that COX-2 protein co-localized with Aß-IR material at the injection site and in endothelial blood vessels, increasing at 72 h. In the Aß oligomer-treated animals, COX-2, IL-1ß, and TNFα proteins were expressed in reactive astrocytes surrounding the injection site and blood vessels at early stages of Aß toxicity. Double-labelling immunofluorescence studies also revealed that GFAP and COX-2 proteins co-localized with NF-κB-positive material at early time-points. In conclusion, our results suggest that in reactive astrocytes and in COX-2 positive cells NF-κB may mediate pro-, and/or inflammatory gene expression and that, develop strategies that target the GFAP/NF-κB and COX-2/NF-κB pathways might contribute to reducing Aß-induced toxicity.

Soluble oligomeric forms of beta-amyloid (Abeta) peptide stimulate Abeta production via astrogliosis in the rat brain.

The aim of this study was to investigate the interaction between beta-amyloid (Abeta) peptide and astrogliosis in early stages of Abeta toxicity. In Wistar rats, anaesthetised with equitesine, a single microinjection of Abeta1-42 oligomers was placed into the retrosplenial cortex. Control animals were injected with Abeta42-1 peptide into the corresponding regions of cerebral cortex. Immunocytochemical analysis revealed an intense Abeta immunoreactivity (IR) at the level of Abeta1-42 injection site, increasing from the first 24 h to later (72 h) time point. Control injection showed a light staining surrounding the injection site. In Abeta oligomers-treated animals, Abeta-immunopositive product also accumulates in cortical cells, particularly in frontal and temporal cortices at an early (24 h) time point. Abeta-IR structures-like diffuse aggregates forms were also observed in hippocampus and in several cortical areas, increasing from the first 24 h to later (72 h) time point. In control animals no specific staining was seen neither in cortical cells nor in structures-like diffuse aggregates forms. Injections of Abeta oligomers also induce activation of astrocytes surrounding and infiltrating the injection site. Astrocyte activation is evidenced by morphological changes and upregulation of glial fibrillary acidic protein (GFAP). By GFAP immunoblotting we detected two immunopositive protein bands, at 50 and 48 kDa molecular mass. Confocal analysis also showed that GFAP co-localized with Abeta-IR material in a time-dependent manner. In conclusion, our results indicate that astrocyte activation might have a critical role in the mechanisms of Abeta-induced neurodegeneration, and that should be further studied as possible targets for therapeutic intervention in AD.

The human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 (VPAC1) promoter: characterization and role in receptor expression during enterocytic differentiation of the colon cancer cell line Caco-2Cl.20.

The basic organization of the human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor (VPAC) 1 promoter was investigated after cloning the 5'-flanking region (1.4 kb) of the VPAC1 gene from a human genomic library. Subsequent functional analysis of various deletions of the 5'-flanking sequence, subcloned upstream of a luciferase reporter gene, was carried out in HT-29 cells. The minimal promoter region identified encompasses the -205/+76 sequence and contains a crucial CCAAT box (-182/-178) and a GC-rich sequence. Moreover a region (-1348/-933) containing a silencer element was identified. We previously showed that the expression of the VPAC1 receptor binding site is strictly dependent upon the enterocytic differentiation of human colon cancer Caco-2 cells [Laburthe, Rousset, Rouyer-Fessard, Couvineau, Chantret, Chevalier and Zweibaum (1987) J. Biol. Chem. 262, 10180-10184]. In the present study we show that VPAC1 mRNA increases dramatically when Caco-2Cl.20 cells differentiate, as measured by RNase protection assays and reverse transcriptase-PCR. A single transcript species of 3 kb is detected in differentiated cells by Northern-blot analysis. Accumulation of VPAC1 receptor mRNA is due to a 5-fold increase of transcription rate (run-on assay) without a change in mRNA half-life (9 h). Stable transfections of various constructs in Caco-2Cl.20 cells and subsequent analysis of reporter gene expression, during the enterocytic differentiation process over 25 days of culture, further indicated that the -254/+76 5'-flanking sequence is endowed with the regulatory element(s) necessary for transcriptional regulation of VPAC1 during differentiation. Altogether, these observations provide the first characterization of the basic organization of the human VPAC1 gene promoter and unravel the crucial role of a short promoter sequence in the strict transcriptional control of VPAC1 expression during differentiation of human colon cancer Caco-2 cells.

Identification of functional somatostatin receptors and G-proteins in a new line of human foetal lung fibroblasts.

A new line (FP) of human foetal lung fibroblasts was analysed for the expression of functional, G-protein coupled somatostatin receptors (SSTR). By means of RT-PCR, we identified the expression of SSTR1, SSTR2, SSTR3 and SSTR4, but not SSTR5, subtypes. The same technical approach evidenced the expression of stimulatory (alphas) and inhibitory (alphai1, alphai2 and alphai3) G-protein subunits. The functionality of SSTR was established from the observation of a dose-dependent inhibitory role of SST upon isoproterenol-stimulated adenylyl cyclase activity, an effect that involves G-protein action. Moreover, the functionality of G-proteins was assessed by means of experiments with forskolin and a nonhydrolysable GTP analogue that showed either Gi or Gs activation in the regulation of adenylyl cyclase. Present results represent a first pharmacological characterization of this new line of human foetal lung fibroblasts. The selective presence of some SSTR subtypes and G-protein subunits in addition to the regulatory network of the adenylyl cyclase pathway are features of recognized involvement in cell growth mechanisms. It is of interest for a cell class widely used to study this topic but also important in lung physiology and pathophysiology.

Semi-automated flow cytometric analysis of CD34-expressing hematopoietic cells in peripheral blood progenitor cell apheresis products.

BACKGROUND: The measurement of CD34+ cells is the most important step in the quality control of peripheral blood progenitor cell apheresis products. For this purpose, flow cytometry is applied. Recently, a new test kit has been introduced for the enumeration of CD34-expressing cells, in combination with software support for semi-automation of data acquisition and analysis. STUDY DESIGN AND METHODS: This study evaluated the ProCOUNT kit. Ninety samples obtained from peripheral blood progenitor cell apheresis products from 39 patients with hemato-oncologic diseases were analyzed. For data acquisition and analysis, ProCOUNT software was used. Data comparison was performed with parallel measurements according to the International Society for Hematotherapy and Graft Engineering (ISHAGE) guidelines and the German reference protocol for analysis of CD34-expressing cells. RESULTS: Correlation of the German and ISHAGE techniques was excellent (r2 = 0.99). The initial correlation coefficient of ProCOUNT analysis with the German protocol was r2 = 0.89. In 21 (23.3%) of 90 ProCOUNT analyses, a warning message was encountered from the ProCOUNT software. Following manual reevaluation of these data with CellQUEST software, a correlation of r2 = 0.96 with the German protocol and r2 = 0.97 with the ISHAGE analyses was obtained. ANOVA testing revealed significant differences between ProCOUNT and ISHAGE techniques (p<0.05) and between ProCOUNT and the German protocol (p<0.05). No statistically significant difference between ISHAGE and German protocol was observed (p = 0.19). CONCLUSION: The ProCOUNT kit and software for semi-automated data acquisition and analysis represents a further step toward standardization of CD34 cell quantitation in peripheral blood progenitor cell apheresis products. However, the occurrence of software warnings is high, and analysis or data reevaluation by experienced staff is still mandatory. Therefore, currently there is no definite advantage of the kit and software over the existing guidelines for CD34+ analysis in peripheral blood progenitor cell grafts.