Peripheral blood lymphocyte subsets in multiple myeloma and monoclonal gammopathy of undetermined significance

Summary. Peripheral blood lymphocyte subsets were analysed by flow cytometry and compared among 43 patients with untreated multiple myeloma (MM), 16 patients with monoclonal gammopathy of undetermined significance (MGUS) and 26 controls. The age and sex distributions of the patients and controls were comparable, which is important, since in the controls there was a significant effect of age and/or sex on the number of CD3⁺, CD57⁺, CD8⁺57⁺, CD16⁺ and CD3^-56⁺ lymphocyte subsets, and on the CD4⁺/CD8⁺ and CD4⁺Leu–8⁺/CD4⁺ ratios. In MM, the number of CD8⁺ and CD57⁺ cells and the CD4⁺/CD8⁺ ratio were related to the clinical stage. The number of CD20⁺, CD3⁺, CD4⁺, CD16⁺ and CD3^-56⁺ cells and the CD3⁺/CD20⁺ ratio were significantly different in MM patients compared to age- and sex-matched controls as was the number of CD3¹ and CD4¹ cells of MGUS patients compared to controls. Further, there were significant differences in the CD3^-/CD20⁺ ratio between MM and MGUS patients and between stage I MM and MGUS. The role of peripheral blood lymphocyte subsets in differentiating monoclonal gammopathies merits further study.     

Reduced numbers of regulatory B cells are negatively correlated with disease activity in patients with new-onset rheumatoid arthritis

This study is aimed at determining the numbers of circulating Treg and Breg cells in patients with new-onset rheumatoid arthritis and during subsequent drug therapies. Patients were treated orally with 10 mg methotrexate weekly, and 20 mg leflunomide and 60 mg common threewingnut root daily (Lei Gong Teng) for 12 weeks, but received no steroid therapy. Basal measurements were performed of serum C-reactive protein, anticyclic citrullinated peptide antibody, and erythrocyte sedimentation rate, and the numbers of cluster of differentiation CD4⁺CD25⁺Foxp3⁺ T cells, interleukin 10 (IL10)-expressing on CD5⁺CD1d⁺ and TIM1⁺ B cells. Compared with the healthy controls, patients exhibited significantly less numbers of circulating CD19⁺TIM1⁺IL10⁺, CD19⁺CD5⁺CD1d⁺IL10⁺ B cells and CD4⁺CD25⁺Foxp3⁺ T cells (P?<?0.001, all). Drug therapy modulated the balance of different subsets of Breg and Treg cells. The numbers of CD19⁺TIM1⁺IL10⁺ and CD19⁺CD5⁺CD1d⁺IL10⁺ B cells correlated positively with the numbers of CD4⁺CD25⁺Foxp3⁺ T cells in these patients (r?=?0.707, P?=?0.001; r?=?0.481, P?=?0.007, respectively). The values of DAS28 were negatively correlated with the numbers of CD19⁺TIM1⁺IL10⁺ and CD19⁺CD5⁺CD1d⁺IL10⁺ B cells, and CD4⁺CD25⁺Foxp3⁺ T cells (r?=??0.533, P?=?0.023; r?=??0.442, P?=?0.016; and r?=??0.444, P?=?0.014, respectively). Of note, TIM1⁺ B cells identified more circulating IL10⁺ B cells than CD5⁺CD1d⁺ B cells. Our data indicate that Breg and Treg cells have a potentially crucial role in controlling disease activity in rheumatoid arthritis patients, and TIM1⁺ Breg cells may be a viable therapeutic target for these patients.     

Mechanism of Ca2+ resistance in adult heart cells isolated with trypsin plus Ca2+

Ca²⁺-resistant heart cells prepared with trypsin and Ca²⁺ leak Na⁺ and K⁺ more slowly than Ca²⁺-susceptible cells prepared without trypsin and Ca²⁺. The two preparations show similar leak rates for amino acids and nucleotides. Cells prepared with Ca²⁺ alone show low ion leak rates, but the yield of rod-shaped cells is less than half that when trypsin is present. Cells prepared with trypsin alone show high ion leak rates. The Na⁺-K⁺ ATPase activity of Ca²⁺-susceptible cells appears to be approximately three-fold greater than that of Ca²⁺-resistant cells. Imposing a Na⁺-K⁺ leak by the addition of gramicidin D causes no stimulation of Na⁺-K⁺ ATPase in Ca²⁺-susceptible cells, but stimulates the activity of Ca²⁺-resistant cells up to that of the Ca²⁺-susceptible cells. Ca²⁺-resistant cells appear to contain more K⁺ and less Na⁺ than Ca²⁺-susceptible cells. Treatment of Ca²⁺-resistant cells with ouabain (1 mm) for 5 min changes the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ balance to approximately that of the Ca²⁺-susceptible cells, and induces a similar degree of Ca²⁺ susceptibility. We therefore conclude that treatment with trypsin plus Ca²⁺ confers Ca²⁺ resistance by keeping the permeability of the sarcolemma to Na⁺ and K⁺ sufficiently low to allow the Na⁺-K⁺ ATPase and $\text{Na}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{+}}$ exchanger to maintain normal gradients of Na⁺, K⁺ and Ca²⁺. The agent responsible for maintaining low ion permeability appears to be Ca²⁺ itself, while trypsin increases the yield and purity of the Ca²⁺-resistant cells.     

Microarray analysis reveals similarity between CD8+CD28- T cells from young and elderly persons, but not of CD8+CD28+ T cells

We isolated highly purified CD8⁺CD28⁺ and CD8⁺CD28⁻ T cell populations from healthy young and elderly persons for gene expression profiling using Affymetrix oligonucleotide microarrays. We demonstrate that the gene expression profile of CD8⁺CD28⁻ T cells is very similar in young and elderly persons. In contrast, CD8⁺CD28⁺ in elderly differ from CD8⁺CD28⁺ in young persons. Hierarchical clustering revealed that CD8⁺CD28⁺ in elderly are located between CD8⁺CD28⁺ in young and CD8⁺CD28⁻ (young and old) T cells regarding their differentiation state. Our study demonstrates a dichotomy of gene expression levels between CD8⁺CD28⁺ T cells in young and elderly persons but a similarity between CD8⁺CD28⁻ T cells in young and elderly persons. As CD8⁺CD28⁺ T cells from elderly and young persons are distinct due to a different composition of the population, these results suggest that the gene expression profile does not depend on chronological age but depends on the differentiation state of the individual cell types.     

Transmission of chloroplast genes in triploid and tetraploid zygospores of Chlamydomonas reinhardtii: Roles of mating-type gene dosage and gametic chloroplast DNA content

Diploid clones homozygous (mt⁺/mt⁺ or mt^-/mt^-) or heterozygous (mt⁺/mt^-; phenotypically mt^-) for the mating-type locus and homoplasmic for a chloroplast marker conferring resistance to an antibiotic were isolated by artificially induced cell fusion or sexual mating. These diploids were crossed with haploid or diploid strains of opposite mating type and carrying another chloroplast marker. The transmission of the chloroplast genes was analyzed in the triploid and tetraploid zygospores in comparison with diploid zygospores used as controls. The transmission was almost exclusively maternal (mt⁺) (>94%) in the crosses mt⁺ × mt^-, mt⁺/mt⁺ × mt^-, and mt⁺/mt⁺ × mt^-/mt^-. The transmission was preferentially maternal (>76%) in the crosses mt⁺ × mt^-/mt^- whereas in the crosses mt⁺ × mt⁺/mt^-, <50% of the zygospores transmitted the chloroplast allele of maternal (mt⁺) origin. The zygospores produced in crosses mt⁺/mt⁺ × mt⁺/mt^- transmitted the alleles from both parents in >60% of cases. The results show that (i) the presence of one mt⁺ allele in the mt⁺/mt^- (phenotypically mt^-) diploid gametes and (ii) the higher amount of chloroplast DNA molecules (input) present in the diploid gametes versus the haploid ones favor the transmission of the chloroplast allele contributed by these gametes. Moreover, because the zygospores issued from crosses mt⁺/mt⁺ × mt^- and mt⁺ × mt⁺/mt^- were genotypically identical mt⁺/mt⁺/mt^-) but behaved very differently in their chloroplast gene transmission, it was concluded that the molecular events leading to preferential elimination of paternal DNA copies must occur before the fusion of nuclei or chloroplasts in the newly formed zygotes.     

Uncommon cases of immature-type CD56+ natural killer (NK)-cell neoplasms, characterized by expression of myeloid antigen of blastic NK-cell lymphoma

Immature-type CD56⁺ natural killer (NK)-cell neoplasms are classified as either myeloid/NK-cell precursor acute leukemia or blastic NK-cell lymphoma. We identified two cases of immature-type CD56⁺ NK-cell neoplasms that were not categorizable as either of these entities. The first case involved a 74-year-old woman presenting with skin eruptions and pancytopenia due to bone marrow necrosis. Skin biopsy specimen revealed CD4⁺, CD7⁻, CD34⁻, CD43⁺, CD56⁺, CD68⁺, muramidase (lysozyme)⁺, and myeloperoxidase (MPO)⁻, and immunophenotyping of peripheral blood showed CD4⁺, CD7⁻, CD13⁺, CD33⁺, CD34⁻, CD43⁺, CD56⁺, cytoplasmic (cy)CD68⁺, CD123⁺, and HLA-DR⁺. The second case involved a 62-year-old man who had bilateral optic nerve tumor and presented with malignant cells in peripheral blood. Cell surface markers of malignant cells showed CD4⁺, CD7⁻, CD13⁺, CD33⁺, CD34⁻, CD43⁺, CD56⁺, cyCD68⁺, and HLA-DR⁺. The phenotypes of tumor cells in both cases were compatible with blastic NK-cell lymphoma, except for the expression of myeloid antigen. Clinical presentations of these cases showed characteristics of both blastic NK-cell lymphoma and myeloid/NK-cell precursor acute leukemia.     

CD4+Foxp3+ T cells,interleukin-35 (IL-35) and IL-10 in systemic lupus erythematosus patients: Relation to disease activity

Aim of the workTo evaluate three subtypes of CD4⁺Foxp3⁺ T cells, interleukin-35 (IL-35) and IL-10 in systemic lupus eryhtematosus (SLE) patients and study their relation to disease activity.Patients and methodsFifty SLE patients were included and divided according to the SLE disease activity index (SLEDAI) into 2 equal groups with activity or in remission. Twenty healthy subjects were included as controls. All subjects underwent flow cytometric analysis of CD4, CD25, CXCR5 and Foxp3 expression on T cells. Serum IL-35 and IL-10 levels were measured by ELISA.ResultsPatients were 46 females and 4 males with a mean age of 38.0 ± 10.0 years, disease duration of 9.2 ± 6.0 years. The mean SLEDAI was 6.8 ± 3.7 in active ones. SLE patients especially those with activity had significantly reduced percents of CD4⁺CD25⁺Foxp3⁺ and CD4⁺CXCR5⁺Foxp3⁺ T cells, but increased percents of CD4⁺CD25⁻Foxp3⁺ T cells. This was accompanied by significant higher levels of serum IL-35 and IL-10 (p < 0.0001). The SLEDAI in active patients significantly correlated with CD4⁺CD25⁻Foxp3⁺ T cell percent, serum IL-35 and IL-10 levels (p < 0.05) and inversely with the CD4⁺CD25⁺Foxp3⁺ and CD4⁺CXCR5⁺Foxp3⁺ T cell percents (p < 0.05). At cut-off values of 3.29% for CD4⁺CD25⁺Foxp3⁺ T cell, 7.62% for CD4⁺CD25⁻Foxp3⁺ T cell, 1.77% for CD4⁺CXCR5⁺Foxp3⁺ T cell, 22.04 pg/ml for IL-35 level and 30.51 pg/ml for serum IL-10 level were found to be highly sensitive and specific for detecting lupus activity.ConclusionCD4⁺Foxp3⁺ T cells, IL-35 and IL-10 showed high sensitivity and specificity for detecting SLE activity and may be considered as potentially promising therapeutic targets.     

Molecular genotyping and frequencies of A 1 , A 2 , B, O 1 and O 2 alleles of the ABO blood group system in a Kuwaiti population

Expression of the highly polymorphic ABO gene cluster is commonly investigated for blood transfusion and analysis, but little information is available for Middle Eastern populations. This study determined the major ABO allele frequency in a Kuwaiti Arab cohort using a multiplex PCR–RFLP technique; 355 unrelated blood donors of phenotype A¹ (46), A² (31), A¹B (6), A²B (4), B (97) and O (171) were genotyped. DNA fragments of 252 (251 for O ¹ ) and 843 (842 for A ² ) bp spanning the two major exons, 6 and 7, of the ABO gene were amplified and digested with HpaII and KpnI. Thirteen different genotypes could be identified when combining the A ¹ , A ² , B, O ¹ and O ² alleles from the digestion patterns: 1 A ¹ A ¹ (0.28%), 6 A ¹ A ² (1.69%), 38 A ¹ O ¹ (10.71%), 1 A ¹ O ² (0.28%), 1 A ² A ² (0.28%), 30 A ² O ¹ (8.45%), 6 A ¹ B (1.69%), 4 A ² B (1.13%), 12 BB (3.38%), 79 BO ¹ (22.25%), 6 BO ² (1.69%), 167 O ¹ O ¹ (47.04%) and 4 O ¹ O ² (1.13%). Two of the combinations (A ² O ² , O ² O ² ) were not found. All genotypes determined were consistent with the serotypes. The frequencies of the five alleles in the Kuwaiti sample population were ABO*A¹ = 0.0746, ABO*A² = 0.0592, ABO*B = 0.1676, ABO*O¹ = 0.6831 and ABO*O² = 0.0155. These results are discussed with reference to gene frequencies reported for other ethnic groups.     

Structure of the Hf nucleus in high rotational states

The values of the moment of inertia of the ground-state band of ¹⁶⁸Hf from J = 0⁺ to 22⁺ are interpreted as involving a revolving cluster with composition increasing steadily from p^10.5n¹⁴ to p^20.5n²⁴, with radius of revolution 6.390 fm. The excited bands from 14⁺ to 34⁺, 21^- to 33^-, and 18^- to 30^- have nearly the same very large moment of inertia. It is interpreted as involving two caps, each consisting of 11 tritons, on opposite sides of the nearly doubly magic sphere p⁵⁰n⁵², the value of the radius of revolution being 6.445 fm.     

Comparative Studies of the in Vivo Kinetics of Simultaneously Injected 111In- and 51Cr-Labelled Human Platelets

The kinetics of simultaneously injected ¹¹¹In- and ⁵¹Cr-labelled platelets have been assessed in 40 subjects, 13 of them thrombocytopenic. 4 platelet survival models were applied. The mean life-time (MLT) of ⁵¹Cr-platelets from non-thrombocytopenic individuals was found to be slightly, but significantly, longer than that of ¹¹¹In-platelets by applying linear and exponential models for data fitting. The in vivo recovery (IVR) of ¹¹¹In-platelets was significantly higher than that of ⁵¹Cr-platelets in this patient group when using all 4 models. In the group of thrombocytopenic patients no statistically significant differences in MLT or IVR were found between ¹¹¹In- and ⁵¹Cr-platelets. However, for each of the 11 ⁵¹Cr-labelled platelet suspensions with the shortest MLT, a longer MLT was observed in the corresponding ¹¹¹In-platelets, a finding probably related to antibody-induced elution of ⁵¹Cr-activity. The same mechanism might be responsible for an increasing ¹¹¹In-/⁵¹Cr-recovery ratio in the early post-injection period. The efficiency of platelet isolation from blood prior to labelling seemed to influence the IVR, inasmuch as the difference in IVR between ¹¹¹In- and ⁵¹Cr-platelets was eliminated in the group where the yield of ¹¹¹In-platelets surpassed that of the ⁵¹Cr-platelets by more than 15 %.     

Clinical Forms of Human Schistosoma mansoni Infection Are Associated with Differential Activation of T-Cell Subsets and Costimulatory Molecules

The current study has compared the activationstatus and the expression of the CD28 molecule oncirculating CD4⁺ and CD8⁺lymphocytes from patients with different clinical formsof schistosomiasis. The data show that patients with acuteschistosomiasis have an increase on the mean percentageof CD4⁺HLA-DR⁺ cells, whereaschronic asymptomatic patients exhibit an increased meanpercentage of CD8⁺HLA-DR⁺ cells. Patients with the hepatosplenic diseaseshowed an increase in bothCD4⁺HLA-DR⁺ andCD8⁺HLA-DR⁺ cells. Despite thehigh levels of CD8⁺HLA-DR⁺ cellsin hepatosplenic patients, they presented a decreased ratio ofCD8⁺CD28⁺/CD8⁺ cells.These findings of a different percentage of circulatingCD8⁺CD28⁺ cells might explain thedifferent in vitro cellular reactivity of asymptomaticand hepatosplenic patients and the defects in the cytokine secretionpatterns reported in individuals with hepatosplenicschistosomiasis.     

Elevated level of circulating CD4+Helios+FoxP3+ cells in primary Sjogren’s syndrome patients

Objective: This study was designed to investigate the expression of regulatory T cells in primary Sjögren’s Syndrome (pSS) and to evaluate the clinical role of CD4?⁺?Helios⁺?FoxP3⁺ cells in pSS patients.

Methods: CD4?⁺?FoxP3^+?T cells in the peripheral blood of 39 pSS patients and 30 healthy controls were measured by flow cytometry and CD25 and Helios expression were also analyzed. The repression ability of CD4?⁺?CD25^hi cells was tested in vitro. Clinical information of pSS patients was retrospectively collected and their correlations with circulating Treg cells were analyzed. Cytokine levels in plasma were measured by ELISA and correlations with Helios⁺?FoxP3^+?cells were also detected.

Results: Circulating FoxP3^+?and Helios⁺?FoxP3^+?cells were elevated in pSS patients compared with controls. The suppression function of CD4?⁺?CD25^hi cells is not different between two groups. There are inverse correlations between Helios⁺?FoxP3^+?percentage and ESR, IgG, IgM and ESSDAI. Anti-SSB^? patients possess higher level of Helios⁺?FoxP3^+?cells than anti-SSB^+?patients. IL-6, IFNγ and IFNα levels were increased in pSS plasma and there were positive correlations between the levels of IFNγ/IFNα and percentage of Helios⁺?FoxP3^+?cells.

Conclusion: Circulating Helios ⁺?FoxP3⁺ cells were elevated in pSS patients and may contribute to suppressing autoimmunity in pSS patients.     

Characterization of lymphoid cells in the blood of healthy adults: Sequential immunological,cytochemical and cytokinetic studies

With a new method, sequential immunological, cytochemical and cytokinetic studies were done on lymphoid cells in the peripheral blood of 12 healthy adults. Every single lymphoid cell could therefore be characterized by the following markers: surface immunoglobulins (sIg); rosetting with sheep red blood cells (E); unspecific acid alpha-naphthyl acetate esterase (ANAE); and ³HdT incorporation. Significantly more E⁺sIg^? cells were ANAE⁺ than E^?sIg⁺ cells (70% vs. 11%). About half of the E⁺sIg^? cells were ANAE⁺. By combining esterase and surface marker analyses eight subpopulations of lymphoid cells could be distinguished. The most common subpopulations were E⁺sIg^? ANAE⁺ and E⁺sIg^?ANAE^? cells (51% and 22% of all lymphoid cells, respectively). Of all ANAE⁺ cells 90% were E⁺, but 64% of all ANAE^? cells were also E⁺. In all individuals a subpopulation of E⁺sIg⁺ cells was found. The esterase pattern of these cells was similar to that of E^?sIg⁺ cells. The overall labeling index of the lymphoid cells examined was ≦ 0.2%.     

Early plasmacytoid dendritic cell leukemia/lymphoma coexpressing myeloid antigenes

Early plasmacytoid dendritic cell (pDC) leukemia/lymphoma has recently been described as a CD4⁺CD56⁺ lineage negative malignancy with characteristic clinical, morphologic, immunophenotypic, and biological features. We present a case of a 72-year-old man who was diagnosed with isolated skin involvement 30 months ago and received numerous chemotherapy cycles that did not prevent three relapses of the disease, the last two involving the bone marrow. The bone marrow was nearly completely infiltrated with small- to medium-sized blasts displaying a high nuclear to cytoplasmic ratio, a cytoplasm with faint basophilia lacking granulations or Auer rods. Small vacuoles surrounding the nucleus were frequently observed. Flow cytometry showed CD4⁺, CD56⁺, CD45⁺, CD38⁺, HLA-DR⁺, CD33⁺, CD123⁺, CD2^–, cyCD3^–, CD7^–, CD10^–, CD11b^–, CD13^–, CD14^–, CD16^–, CD19^–, cyCD22^–, CD24^–, CD34^–, CD57^–, CD61^–, CD64^–, CD65^–, cyCD79a^–, CD117^–, MPO^–, and TdT^– population. At the second bone marrow relapse, CD117 was also positive. Our patient was initially treated with acute myeloid leukemia-type chemotherapy, later he was given acute lymphoblastic leukemia-type treatment, and at the last relapse he received CHOP chemotherapy. Each treatment led to rapid response of tumor manifestations with disease-free intervals of 7 months, 9 months, and 8 months, respectively. Although patients usually have an ominous prognosis, with only 25% living more than 24 months, our patient is alive after 30+ months and has again achieved complete remission after the last chemotherapy.     

Immunohistochemical identification of lymphocyte subsets and accessory cells in human hyperplastic lymph nodes The functional significance of the compartmentalization of lymphoid tissue

Biopsies from 21 lymph nodes with benign hyperplasia were examined by immuno-enzymatic labelling of frozen sections with a panel of monoclonal antibodies. B-cells (B1⁺, HLA-DR⁺, C3b receptor^+/-) localized in primary follicles, secondary follicles, and areas adjacent to the subcapsular sinus. The B-cells in primary follicles and mantle zones of secondary follicles were indistinguishable (SmIgD⁺, SmIgM⁺, Cylg⁻, T10⁻, CALLA⁻). B-cells adjacent to the subcapsular sinus demonstrated a higher density of SmIgM, and a weaker expression of SmIgD. The germinal centre cells showed a more differentiated phenotype (SmIgD⁻, SmIgM⁺, CyIgM^+/-), and also expressed T10 and CALLA. T-cells (Lyt3⁺Lyt2⁺, Leu4⁺, OKT6⁻, OKT10⁻) localized in paracortial and interfollicular areas, and demonstrated a relative predominance of T-helper/inducer cells (Leu3⁺). T-helper/inducer cells were also identified in secondary follicles. The B-cell areas contained dendritic reticulum cells (R4/23⁺, C3b-receptor⁺). Interdigitating reticulum cells (HLA-DR⁺, OKT6^+/-) localized in T-cell regions. The cells in sinuses demonstrated monocyte/macrophage properties (MO2⁺, Ig⁺, C3b-receptor⁺, HLA-DR^+/-).     

Ultrahigh resolution protein structures using NMR chemical shift tensors

NMR chemical shift tensors (CSTs) in proteins, as well as their orientations, represent an important new restraint class for protein structure refinement and determination. Here, we present the first determination of both CST magnitudes and orientations for ¹³Cα and ¹⁵N (peptide backbone) groups in a protein, the β1 IgG binding domain of protein G from Streptococcus spp., GB1. Site-specific ¹³Cα and ¹⁵N CSTs were measured using synchronously evolved recoupling experiments in which ¹³C and ¹⁵N tensors were projected onto the ¹H-¹³C and ¹H-¹⁵N vectors, respectively, and onto the ¹⁵N-¹³C vector in the case of ¹³Cα. The orientations of the ¹³Cα CSTs to the ¹H-¹³C and ¹³C-¹⁵N vectors agreed well with the results of ab initio calculations, with an rmsd of approximately 8°. In addition, the measured ¹⁵N tensors exhibited larger reduced anisotropies in α-helical versus β-sheet regions, with very limited variation (18 ± 4°) in the orientation of the z-axis of the ¹⁵N CST with respect to the ¹H-¹⁵N vector. Incorporation of the ¹³Cα CST restraints into structure calculations, in combination with isotropic chemical shifts, transferred echo double resonance ¹³C-¹⁵N distances and vector angle restraints, improved the backbone rmsd to 0.16 Å (PDB ID code 2LGI) and is consistent with existing X-ray structures (0.51 Å agreement with PDB ID code 2QMT). These results demonstrate that chemical shift tensors have considerable utility in protein structure refinement, with the best structures comparable to 1.0-Å crystal structures, based upon empirical metrics such as Ramachandran geometries and χ¹/χ² distributions, providing solid-state NMR with a powerful tool for de novo structure determination.     

The Prognostic Role of Peripheral Lymphocyte Subsets in Patients With Acute Pancreatitis

Background

The prognostic role of peripheral lymphocyte subsets in early stage of acute pancreatitis (AP) is unknown.

The Physiology of the Transplanted Small Bowel: An Overview with Insight into Graft Function

Nilsson J, Sjödin L, Gylfe E. Supramaximal inhibition of cholecystokinin-induced pancreatic amylase release involves desensitization to cytoplasmic Ca²⁺. Scand J Gastroenterol 1994;29:561-568.

Background: Cholecystokinin (CCK) is a major stimulant of pancreatic enzyme secretion. The dose-response relationship for CCK-induced secretion is bell-shaped, with a characteristic supramaximal inhibition. The mechanism for this inhibition has now been studied.

Methods: The kinetics of amylase release and the changes of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]i) were recorded during stimulation of guinea-pig pancreatic acinar cells with different concentrations of cholecystokinin octapeptide (CCK-8) and the Ca²⁺ ionophore ionomycin. Results: Individual cells reacted with [Ca²⁺]_i oscillations at 10^?11 10^?10 M CCK-8 and with an initial peak followed by a sustained suprabasal level at 10 ^?9–10^?8 M of the agonist. The latter response was also seen in suspensions of acinar cells at all tested concentrations of CCK-8 and at 10^?6-10^?5 M of ionomycin. With increases of extracellular Ca²⁺ from 0.5 to 5.0 mM there was a rise of [Ca²⁺]_i during exposure to 10^?9-10^?8 M CCK-8 or 10^?5 M ionomycin but a paradoxical decrease at lower concentrations of CCK-8 or ionomycin. A dose-dependent increase of amylase release was seen at CCK-8 concentrations from 10^?11 to 10^?9M. At 10^?9-10^?8M CCK-8 secretion was characterized by an initial peak followed by a sustained phase. Whereas the initial peak of secretion remained unaffected by increasing CCK-8 from 10^?9 to 10^?8M, the sustained phase was inhibited (supramaximal inhibition). Increasing extracellular Ca²⁺ from 0.5 to 5.0 mM transiently enhanced secretion in response to 10^?9 M but lacked effect during supramaximal inhibition of secretion by 10^?8 M CCK-8.

Conclusions: Both initial and sustained CCK-8-stimulated amylase release increase with [Ca²⁺]_i. However, supramaximal inhibition of secretion was not due to a decrease of [Ca²⁺]_i but was characterized by desensitization to the stimulatory effect of [Ca²⁺]_i.     

Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta

Summary Despite the important physiological role of insulin in the regulation of ionic homeostasis, primarily mediated by the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter, the intracellular signalling molecules mediating this effect of insulin have not been elucidated. Treatment of 3T3-L1 fibroblasts with insulin increased total ⁸⁶Rb⁺ (K⁺) uptake from 0.8 ± 0.04 to 1.02 ± 0.05 nmol · mg^–1· protein^–1· min^–1 (p < 0.005). These changes in K⁺ flux, though small, can alter the membrane potential. Uptake occurred through both the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter and both were stimulated by insulin. Interestingly, when bumetanide was used to inhibit the Na⁺/K⁺/2Cl^– cotransporter prior to insulin action, no increase in ⁸⁶Rb⁺ uptake via the Na⁺/K⁺-ATPase was observed. The structurally distinct phosphatidylinositol 3-kinase inhibitors wortmannin (50–200 nmol/l) and LY294 002 (50 μmol/l) attenuated both total insulin-stimulated ⁸⁶Rb⁺ uptake as well as uptake via the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter. Neither the inhibitor of p70 S6 kinase activation, rapamycin (30 ng/ml) nor the mitogen activated protein kinase kinase inhibitor, PD098 059 (50 μmol/l), had any effect on insulin's stimulation of K⁺ influx. A 10 μmol/l concentration of the protein kinase C (PKC) inhibitor bisindolylmaleimide attenuated insulin action but at 1 μmol/l it was ineffective, suggesting involvement of the atypical PKC-ζ isoform. We conclude that insulin-stimulated K⁺ uptake in 3T3-L1 fibroblasts appears to involve concerted regulation of both the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter and we show for the first time that this process is signalled via a pathway involving phosphatidylinositol 3-kinase and PKC-ζ. [Diabetologia (1998) 41: 1199–1204] Received: 20 March 1998 and in revised form: 2 June 1998