Synthesis of site-specific Fab-drug conjugates using ADP-ribosyl cyclases.

Targeted delivery of small-molecule drugs via covalent attachments to monoclonal antibodies has proved successful in clinic. For this purpose, full-length antibodies are mainly used as drug-carrying vehicles. Despite their flexible conjugation sites and versatile biological activities, intact immunoglobulins with conjugated drugs, which feature relatively large molecular weights, tend to have restricted tissue distribution and penetration and low fractions of payloads. Linking small-molecule therapeutics to other formats of antibody may lead to conjugates with optimal properties. Here, we designed and synthesized ADP-ribosyl cyclase-enabled fragment antigen-binding (Fab) drug conjugates (ARC-FDCs) by utilizing CD38 catalytic activity. Through rapidly forming a stable covalent bond with a nicotinamide adenine dinucleotide (NAD+ )-based drug linker at its active site, CD38 genetically fused with Fab mediates robust site-specific drug conjugations via enzymatic reactions. Generated ARC-FDCs with defined drug-to-Fab ratios display potent and antigen-dependent cytotoxicity against breast cancer cells. This work demonstrates a new strategy for developing site-specific FDCs. It may be applicable to different antibody scaffolds for therapeutic conjugations, leading to novel targeted agents.

Targeting CD38/ ADP-ribosyl cyclase as a novel therapeutic strategy for identification of three potent agonists for leukopenia treatment.

Leukopenia is the most common side effect of chemotherapy and radiotherapy. It potentially deteriorates into a life-threatening complication in cancer patients. Despite several agents being approved for clinical administration, there are still high incidences of pathogen-related disease due to a lack of functional immune cells. ADP-ribosyl cyclase of CD38 displays a regulatory effect on leukopoiesis and the immune system. To explore whether the ADP-ribosyl cyclase was a potential therapeutic target of leukopenia. We established a drug screening model based on an ADP-ribosyl cyclase-based pharmacophore generation algorithm and discovered three novel ADP-ribosyl cyclase agonists: ziyuglycoside II (ZGSII), brevifolincarboxylic acid (BA), and 3,4-dihydroxy-5-methoxybenzoic acid (DMA). Then, in vitro experiments demonstrated that these three natural compounds significantly promoted myeloid differentiation and antibacterial activity in NB4 cells. In vivo, experiments confirmed that the compounds also stimulated the recovery of leukocytes in irradiation-induced mice and zebrafish. The mechanism was investigated by network pharmacology, and the top 12 biological processes and the top 20 signaling pathways were obtained by intersecting target genes among ZGSII, BA, DMA, and leukopenia. The potential signaling molecules involved were further explored through experiments. Finally, the ADP-ribosyl cyclase agonists (ZGSII, BA, and DMA) has been found to regenerate microbicidal myeloid cells to effectively ameliorate leukopenia-associated infection by activating CD38/ADP-ribosyl cyclase-Ca2+-NFAT. In summary, this study constructs a drug screening model to discover active compounds against leukopenia, reveals the critical roles of ADP-ribosyl cyclase in promoting myeloid differentiation and the immune response, and provides a promising strategy for the treatment of radiation-induced leukopenia.

PD-1 and PD-L1 gene expressions and their association with Epstein-Barr virus infection in chronic lymphocytic leukemia

PurposeThe PD-1 (programmed cell death-1) receptor is expressed on the surface of activated T cells. Its ligand, programmed cell death ligand-1 (PD-L1), is expressed on the surface of dendritic cells or macrophages. The PD-1/PD-L1 interaction ensures prevention of autoimmunity by activating the immune system only when needed. In cancers, PD-L1 expressed on the tumour cells binds to PD-1 receptors on the activated T cells, leading to inhibition of the cytotoxic T cells and immunosuppression. PD-1/PD-L1 pathway is upregulated in EBV infection that is known to worsen the CLL prognosis. Therefore, we aimed to study the association between PD-1 and PD-L1 expressions, EBV status and the CLL prognosis.Methods and patientsThe study was conducted on 80 newly diagnosed CLL patients and 80 controls. We analyzed PD-1 and PD-L1 expressions and EBV-DNA load by real-time PCR. The cytogenetic abnormalities and expression of ZAP70 and CD38 were detected by FISH and Flow cytometry, respectively.ResultsPD-1/PD-L1 expressions were significantly upregulated in CLL patients compared to controls. In addition, their mRNA levels were significantly higher in EBV( +) versus EBV( −) patients. High expression of PD-1/PD-L1 was associated with poor prognostic markers (RAI stages of CLL, del 17p13, ZAP70, and CD38 expression), failure of complete remission, shorter progression-free survival, and overall survival.ConclusionHigh expression of PD-1 and PD-L1, together with high EBD-DNA load were linked to worse prognosis in CLL. In addition, PD-1 and PD-L1 might represent suitable therapeutic targets for patients suffering from aggressive CLL. (AU)

Associação entre diabéticos e câncer intestinal com o risco de mutação no gene CD38 na população iraniana / Association between diabetics and intestinal cancer with the risk of mutation in cd38 gene in iranian population

ABSTRACT BACKGROUND: Intestinal cancer often occurs in type 2 diabetic patients. The concept of increasing insulin levels and insulin-like growth factor in the blood with type 2 diabetes are stimulated with the growth and depletion of cloned cell walls, and the continuation of this process leads to the cellular deformation. This is the evidence for intestinal cancer in type 2 diabetes in population. OBJECTIVE: In this study, we aimed to find out the relationship between diabetics and intestinal cancer based on CD38 gene mutation. METHODS: Samples were collected from 200 population including normal and case ones. PCR products related to rs 6449181 of CD38 gene was amplified with ARMS-PCR technique, and a 420-bp sharp banding was observed as well. According three ARMS-PCR techniques, three primers were designed by oligo7 software. Primers include F1, F2 and R (amplifying for normal, mutant and reverse primer respectively). RESULTS: This band was observed using a primer F1 that carries the wild type nucleotide using a primer, and when it is used with the F2 primer, it brings the mutant primer to populations of patients with diabetes and diabetes-cancer. In addition, the clinical results including body mass index, blood glucose and insulin level were analyzed. The means ±SD and Tuckey's post hoc test were significant between the clinical characterization parameters between cases and healthy populations. The allelic gene frequencies and Hardy-Weinberg equilibrium between nucleotides were evaluated, and the significant level between the alleles and gene frequencies was observed. CONCLUSION: In general, the current study found that there is a relationship between diabetes and intestinal cancer among the studied populations.

RESUMO CONTEXTO: O câncer intestinal ocorre frequentemente em pacientes diabéticos tipo 2. O conceito que aumento dos níveis de insulina e fator de crescimento semelhante à insulina no sangue com diabetes tipo 2 sejam estimulados com o crescimento e esgotamento das paredes celulares clonadas, e a continuação desse processo levaria à deformação celular. Esta é a evidência para câncer intestinal em diabetes tipo 2 na população. OBJETIVO: Neste estudo, buscou-se descobrir a relação entre diabéticos e câncer intestinal com base na mutação genética CD38. MÉTODOS: Foram coletadas amostras de duzentos habitantes, incluindo os normais e os casos. Produtos PCR relacionados ao rs 6449181do gene CD38 foi amplificado com a técnica ARMS-PCR, e uma banda afiada de 420 bp também foi observada. De acordo com três técnicas ARMS-PCR, três primers foram projetados pelo software Oligo7. Os primers incluem F1, F2 e R (amplificando para primer normal, mutante e reverso, respectivamente). RESULTADOS: Esta banda foi observada usando um primer F1 que carrega o nucleotídeo do tipo selvagem usando um primer e quando é usado com o primer F2, ele traz o primer mutante para populações de pacientes com diabetes e diabetes-câncer. Além disso, foram analisados os resultados clínicos, incluindo índice de massa corporal, glicemia e nível de insulina. As médias ±SD e Tuckey's post hoc test foram significativas entre os parâmetros de caracterização clínica entre os casos e populações saudáveis. Foram avaliadas as frequências genéticas alélicas e o equilíbrio de Hardy-Weinberg entre nucleotídeos e observou-se o nível significativo entre os alelos e as frequências genéticas. CONCLUSÃO: Em geral, o presente estudo constatou que há relação entre diabetes e câncer intestinal entre as populações estudadas.

Lack of association between three single nucleotide polymorphisms in the PARK9, PARK15, and BST1 genes and Parkinson's disease in the northern Han Chinese population / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Parkinson's disease (PD) is an autosomally inherited neurodegenerative disease in elderly people. The etiology of PD has long been thought to be associated with both genetic and environmental factors. To explore potential genetic risk factors for PD in the northern Han Chinese population, we investigated three single nucleotide polymorphisms (SNPs) (rs4538475, rs11107 and rs12564040) in the BST1, PARK15 and PARK9 genes.</p><p><b>METHODS</b>Genomic DNA from 215 PD patients and 212 matched controls was amplified in two independent PCR systems and subsequently genotyped by digestion with the endonuclease PstI. Genetic parameter and association studies were carried out with SPSS 13.0 and PLINK 1.07 software.</p><p><b>RESULTS</b>We could accurately detect all genotypes in the three loci with the PCR-RFLP or mismatched PCR-RFLP techniques. The observed heterozygosities of the rs4538475 and rs11107 loci in PD and control groups ranged from 0.460 - 0.481 and 0.410 - 0.441, in BST1, PARK15 respectively, while we detected no heterozygosity at the rs12564040 locus in PARK9. The similar distributions of genotypic frequency between both groups suggest that the three SNPs investigated in this study are unlikely to play roles as common risk factors or pathogenic mutations for PD in northern Han Chinese.</p><p><b>CONCLUSION</b>The SNPs investigated in the BST1, PARK15 and PARK9 genes associated with PD susceptibility are not associated with PD in the northern Han Chinese population.</p>

Discovery of a small-molecule inhibitor for kidney ADP-ribosyl cyclase: Implication for intracellular calcium signal mediated by cyclic ADP-ribose

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger, cyclic ADP- ribose (cADPR), from beta-NAD+. A prototype of mammalian ADPR-cyclases is a lymphocyte antigen CD38. Accumulating evidence indicates that ADPR-cyclases other than CD38 are expressed in various cells and organs. In this study, we discovered a small molecule inhibitor of kidney ADPR-cyclase. This compound inhibited kidney ADPR-cyclase activity but not CD38, spleen, heart or brain ADPR-cyclase activity in vitro. Characterization of the compound in a cell-based system revealed that an extracellular calcium-sensing receptor (CaSR)- mediated cADPR production and a later long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse mesangial cells were inhibited by the pre-treatment with this compound. In contrast, the compound did not block CD3/TCR-induced cADPR production and the increase of [Ca2+]i in Jurkat T cells, which express CD38 exclusively. The long-lasting Ca2+ signal generated by both receptors was inhibited by pre-treatment with an antagonistic cADPR derivative, 8-Br-cADPR, indicating that the Ca2+ signal is mediated by the ADPR-cyclse metabolite, cADPR. Moreover, among structurally similar compounds tested, the compound inhibited most potently the cADPR production and Ca2+ signal induced by CaSR. These findings provide evidence for existence of a distinct ADPR-cyclase in the kidney and basis for the development of tissue specific inhibitors.

Changes in NAD/ADP-ribose metabolism in rectal cancer

The extent of ADP-ribosylation in rectal cancer was compared to that of the corresponding normal rectal tissue. Twenty rectal tissue fragments were collected during surgery from patients diagnosed as having rectal cancer on the basis of pathology results. The levels of ADP-ribosylation in rectum cancer tissue samples (95.9 ± 22.1 nmol/ml) was significantly higher than in normal tissues (11.4 ± 4 nmol/ml). The level of NAD+ glycohydrolase and ADP-ribosyl cyclase activities in rectal cancer and normal tissue samples were measured. Cancer tissues had significantly higher NAD+ glycohydrolase and ADP-ribosyl cyclase activities than the control tissues (43.3 ± 9.1 vs 29.2 ± 5.2 and 6.2 ± 1.6 vs 1.6 ± 0.4 nmol mg-1 min-1). Approximately 75 percent of the NAD+ concentration was consumed as substrate in rectal cancer, with changes in NAD+/ADP-ribose metabolism being observed. When [14C]-ADP-ribosylated tissue samples were subjected to SDS-PAGE, autoradiographic analysis revealed that several proteins were ADP-ribosylated in rectum tissue. Notably, the radiolabeling of a 113-kDa protein was remarkably greater than that in control tissues. Poly(ADP)-ribosylation of the 113-kDa protein in rectum cancer tissues might be enhanced with its proliferative activity, and poly(ADP)-ribosylation of the same protein in rectum cancer patients might be an indicator of tumor diagnosis.

Virological and immunological outcomes in HIV-1-infected Chinese patients treated with a combination of Efavirenz and Indinavir for 48 weeks / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The incidence of HIV-1-related infection diseases and the mortality of AIDS have dramatically decreased since highly active antiretroviral therapy began to be used clinically in China in 1999. And we initiated a second clinical trial using a combination of Efavirenz and Indinavir to observe the effects of the immunoreaction.</p><p><b>METHODS</b>Twenty patients with laboratory-confirmed chronic HIV-1 infection were recruited. Blood samples were collected initially and during the weeks after initiation of treatment. Within 48 hours of blood sampling, peripheral blood plasma and mononuclear cells were separated using routine methods. HIV-1 viral load was measured in thawed plasma samples. Within 48 hours of peripheral blood sampling, CD4(+) and CD8(+) T cell subsets were enumerated.</p><p><b>RESULTS</b>The drug regimen was efficient in reducing HIV-1 plasma viral load and increasing total CD4(+) T cell counts. The percentage of CD4(+) and CD8(+) T cell subsets expressing CD38 and HLA-DR activation markers was positively correlated with plasma viral load and tended to normalize.</p><p><b>CONCLUSIONS</b>The combination of Efavirenz and Indinavir was generally well tolerated and efficient at reducing HIV-1 RNA. Furthermore, the treatment improved the immunological function.</p>

Stem Cells Expressing Homing Receptors Could be Expanded From Cryopreserved and Unselected Cord Blood

We assessed the cytokine combinations that are best for ex vivo expansion of cord blood (CB) and the increment for cell numbers of nucleated cells, as well as stem cells expressing homing receptors, by an ex vivo expansion of cryopreserved and unselected CB. Frozen leukocyte concentrates (LC) from CB were thawed and cultured at a concentration of 1x10(5)/mL in media supplemented with a combination of SCF (20 ng/mL)+TPO (50 ng/mL)+FL (50 ng/mL)+/-IL-6 (20 ng/mL)+/-G-CSF (20 ng/mL). After culturing for 14 days, the expansion folds of cell numbers were as follows: TNC 22.3+/-7.8~26.3+/-4.9, CFU-GM 4.7+/-5.1~11.7+/-2.6, CD34+CD38- cell 214.0+/-251.9~464.1+/-566.1, CD34+CXCR4+ cell 4384.5+/-1664.7~7087.2+/-4669.3, CD34+VLA4+ cell 1444.3+/-1264.0~2074.9+/-1537.0, CD34+VLA5+ cell 86.2+/-50.9~ 113.2+/-57.1. These results revealed that the number of stem cells expressing homing receptors could be increased by an ex vivo expansion of cryopreserved and unselected CB using 3 cytokines (SCF, TPO, FL) only. Further in vivo studies regarding the engraftment after expansion of the nucleated cells, as well as the stem cells expressing homing receptors will be required.

Effect of Tpo and/or IL-11 gene modified stromal cells on the expansion of CD34+ CD38- hematopoietic primitive progenitor cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Tpo and/or IL-11 gene modified stromal cells on the expansion of CD(34)(+) hematopoietic stem/progenitor cells in cord blood.</p><p><b>METHODS</b>Retroviral vectors containing Tpo or IL-11 gene were constructed and used to transfect the stromal cell line HFCL. Tpo and/or IL-11 mRNA was assayed by Northern blot. Non-modified stromal cells were used, CD(34)(+) hematopoietic stem/progenitor cells from cord blood were expanded on gene-modified stromal cells for 7 days. The phenotype of CD(34)(+)CD(38)(-) primitive progenitors was detected by flow cytometry.</p><p><b>RESULTS</b>HFCL expressed Tpo and/or IL-11 mRNA after transfected by the retroviral vectors. The percentages of CD(34)(+)CD(38)(-) primitive progenitors in the cultures of Tpo, IL-11 and Tpo + IL-11 modified HFCL were (1.8 +/- 0.24)%, (1.62 +/- 0.23)%, and (2.45 +/- 0.28)%, respectively, which were higher than that in the control [(0.8 +/- 0.23)%].</p><p><b>CONCLUSION</b>The stromal cells modified by Tpo and/or IL-11 gene were able to enhance ex vivo expansion of CD(34)(+) and CD(34)(+)CD(38)(-) hematopoietic stem/progenitor cells from cord blood.</p>

Effect of the number of CD34+CD38+ cells in cord blood on hematopoietic reconstitution in patients with acute leukemia after umbilical cord blood transplantation / 中国实验血液学杂志

The objective of this research was to explore whether the number of CD34(+)CD38(+) cells infused affects hematopoietic reconstitution after cord blood transplantation. The number of CD34(+)CD38(+) cells in cord blood was analysed with flow cytometry after freezethawing. The body weight and time for neutrophil and platelet recovery were measured in 20 children with acute leukemia. The results showed that the median number of CD34(+)CD38(+) cells infused was 29.47 (9.85 - 325.71) x 10(4)/kg. A median time for neutrophil recovery (> 5 x 10(8)/L) in 20 patients was 18.5 (11 - 32) days, and time for platlet recovery (> 2 x 10(10)/L) in 19 of 20 patients was 45 (12 - 118) days. The number of CD34(+)CD38(+) cells infused correlated with time to neutrophil and platelet recovery (r = -0.577, P < 0.01 and r = 0.503, P < 0.05, respectively). In conclusion, the number of CD34(+)CD38(+) cells infused is correlated with the time for hematologic recovery.

Immunophenotypic characteristics of multiple myeloma cells / 中国实验血液学杂志

To investigate the immunophenotypic characteristics of multiple myeloma (MM) cells, 20 bone marrow samples from patients with multiple myeloma were analyzed by flow cytometry with three-color direct immunofluorescence staining. Results showed that all of myeloma cells expressed bright CD38, dim or negative CD45 and negative CD19. Most of the cells were CD56(+) and a small portions were ckappa(+) or clambda(+), or CD20(+). The phenotypes of normal plasmocyte, CD19(+) and CD56(-), except CD56(-) in one-third samples, were not appeared in all detected samples. It was concluded that the surface marker analysis of myeloma cells is a useful tool for diagnosis and further evaluating prognosis of multiple myeloma.

Comparative study on activated immunocytes of human bone marrow and peripheral blood by cytokines / 中国实验血液学杂志

To study immunophenotype and cytotoxicity of the immunocytes in bone marrow and peripheral blood after activation by combined cytokines, mononuclear cells (MNC) of bone marrow and peripheral blood were activated by IFN-gamma, IL-1, IL-2 and McAb-CD3 in vitro. The cell amount and morphology during culture were observed. Cytochemical staining and immunophenotype analysis were done before and after culture in two groups of MNC. Cytotoxicity was tested by MTT method. The results showed that the cell number of two groups increased obviously in culture (P < 0.05), while the peripheral blood mononuclear cells increased more markedly (P < 0.05). The cytochemical staining showed POX decrease, but PAS increase in two groups. The positive ratios of CD3(+), CD56(+) and CD38(+) cells in two groups increased obviously after culture (P < 0.05), but there was no significant difference between those two groups. CD3(+) CD56(+) cells increased obviously in peripheral blood mononuclear cells activated by cytokines (P < 0.05), but CD3(+) CD56(+) cells did not increase in bone marrow mononuclear cells. There was no significant difference between two groups' cytotoxicity. It was concluded that IFN-gamma, IL-1, IL-2 and McAb-C D3 increased cell number and cytotoxicity of both bone marrow and peripheral blood mononuclear cells that can be used in cell immunotherapy.

Initial division behavior of cord blood hematopoietic stem cells depends on microenvironment / 中华血液学杂志

<p><b>OBJECTIVE</b>The influence of the mimic hematopoietic microenvironment and adhesion factor on the initial divisional behavior of human cord blood hematopoietic progenitors was studied in the culture system with certain cytokines.</p><p><b>METHODS</b>(1) CD(34)(+) CD(38)(-) single cell was sorted by FACS. (2) The stem cell supporting stromal feeder layer AFT024 and single adhesive factor fibronectin (Fn) were used in the culture system and their influence on the initial division was observed.</p><p><b>RESULTS</b>(1) In the presence of the combined cytokines, the CD(34)(+) CD(38)(-) human cord blood cells displayed fixed fraction of quiescent, slow and fast division, and asymmetric division. (2) There was no influence of adhesive factor itself on initial division of CD(34)(+) CD(38)(-) cells. (3) The hematopoietic microenvironment mimicked by AFT024 promoted CD(34)(+) CD(38)(-) cells to proliferate extensively and undergo more asymmetric division.</p><p><b>CONCLUSIONS</b>(1) CD(34)(+) CD(38)(-) cells are heterogeneous and composed of various subpopulations with different initial proliferative behavior, including asymmetric division. (2) The hematopoietic microenvironmental mimicked by AFT024 supports the hematopoietic progenitors better than cytokines and single adhesion factor do, for their proliferation extensively and preservation the self-renewal capacity.</p>

Synergic effect of heparan sulfate and cytokines on in vitro growth of human umbilical cord blood hematopoietic cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the synergic effect of heparan sulfate (HS) and cytokines on the growth of human umbilical cord blood (UCB) hematopoietic cells.</p><p><b>METHODS</b>Hematopoietic cells from human UCB were cultured with (1) cytokines (rhIL-3, rhIL-1beta, rhIL-6 and SCF) or (2) SN (supernatant from cultured human marrow stromal cells) and cytokines, or (3) SN, or (4) HS and cytokines. Cellular proliferation, CFU-GM yields and changes of cell immunophenotype were observed.</p><p><b>RESULTS</b>Hematopoietic cell proliferation reached peak at the 14th day. The number of total nucleated cells increased 134.5-, 171.3-, 81.5- and 167.2-fold in (1), (2), (3) and (4) groups, respectively, at the 21th day, the (3) group significantly decreased. CD(34)(+) cells increased at the 7th day and reached peak, at the 14th day with a percentage of 68.4%, 82.5%, 69.8% and 79.3%, and at the 21th day 56.2%, 71.7.%, 12.3% and 73.3%, respectively. CD(33)(+) cells reached peak at the 14th day and increased by 80.2%, 68.6%, 81.6% and 70.3%, respectively, and remained these levels at the 21th day. CD(38)(+) cells increased by 66.6%, 73.8%, 70.4% and 71.9% at the 7th day and remained this level at the 14th and the 21th day. From the first week of culture, the percentage of CFU-GM increased in all of the four groups, at the second week of culture, it increased by 250%, 279%, 217% and 273%, and at the third week still increased by 151%, 240%, 145% and 231%, respectively.</p><p><b>CONCLUSION</b>The combination of SN and cytokines have a synergic effect in promoting proliferation of hematopoietic cell from human UCB. The synergic effect remained the same when SN was replaced by heparan sulfate.</p>

Study of lymphocyte subsets and its activated-molecules in patients with paroxysmal nocturnal hemoglobinuria / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between subsets of lymphocytes and between its activated status and the clinical manifestations in patients with PNH, and to unfold immunological mechanism in the pathogenesis of PNH.</p><p><b>METHODS</b>The peripheral blood mononuclear cells (PBMNC) from 18 PNH patients and 20 controls were separated into two subpopulations using anti-CD(59) monoclonal antibody combined with goat-anti-mouse IgG immunomagnetic beads. CD(3)(+), CD(4)(+) and CD(8)(+) lymphocyte subsets were detected by flow cytometry. In 6 newly diagnosed patients, phenotypes associated with T cell activation such as CD(28)(+)/CD(4)(+) or CD(8)(+) cells, CD(8)(+) CD(38)(+) cells, and HLA-DR(+)/CD(4)(+) or CD(8)(+), and NK (CD(3)(-) CD(16)(+)) cells were detected in the peripheral blood.</p><p><b>RESULT</b>Patients with PNH showed significantly increased CD(3)(+) CD(8)(+)/CD(3)(+) CD(4)(+) ratio as compared with controls (1.22 +/- 0.51 vs 0.86 +/- 0.27, P < 0.05), and the CD(3)(+) CD(8)(+)/CD(3)(+) CD(4)(+) ratio in CD(59)(-) PBMC was higher than that in CD(59)(+) PBMC (2.31 +/- 1.56 vs 0.62 +/- 0.27, P < 0.05). The ratios of CD(4)(+) CD(28)(+)/CD(4)(+) markedly decreased and CD(8)(+)HLA-DR(+)/CD(8)(+) increased.</p><p><b>CONCLUSION</b>Patients with PNH appear to have abnormalities in their lymphocytes. Increased ratios of CD(3)(+) CD(8)(+)/CD(3)(+) CD(4)(+) and HLA-DR(+) CD(8)(+)/CD(8)(+) lymphocytes as well as declined ratio of CD(4)(+) CD(28)(+)/CD(4)(+) lymphocytes might be involved in the pathogenesis of PNH.</p>