Locomotion of T cells from patients with cutaneous T-cell lymphoma (Sezary syndrome and mycosis fungoides)

Peripheral blood T cells from eight patients with cutaneous lymphoma (four each with Sezary syndrome or mycosis fungoides) and T cells from skin tumor of one patient each with Sezary syndrome or mycosis fungoides were studied for their locomotor responses to the chemoattractant, casein. Nonmalignant peripheral blood T cells from each patient with mycosis fungoides moved normally. Malignant T cells from skin tumor of patients with mycosis fungoides or Sezary syndrome did not move in the presence of casein. Peripheral blood malignant T cells (Sezary cells) from three of four patients with Sezary syndrome either moved very poorly or did not move at all. The circulating Sezary cells from the fourth patient with Sezary syndrome responded moderately to the chemoattractant, casein. Two of three patients with Sezary syndrome with poor or no locomotor response of T cells underwent therapeutic leukopheresis without any demonstrable effect on their skin infiltration. The patient whose malignant T cells demonstrated moderate locomotor response to casein had a leukemic blast crisis and at that time her skin became free of malignant cells. A repeat study of her circulating T cells at that time demonstrated almost normal locomotor response to casein. These results demonstrate that the locomotor properties of malignant T cells in patients with Sezary syndrome may have prognostic significance.     

Analysis of PI (phosphatidylinositol)-anchoring antigens in a patient of paroxysmal nocturnal hemoglobinuria (PNH) reveals deficiency of 1F5 antigen (CD59), a new complement-regulatory factor.

FACS analysis together with PIPLC treatment was applied to PI-anchoring antigens such as DAF (decay-accerelating factor, CD55), 1F5 antigen (CD59), CD14 and CD16 on the cell surfaces of blood cells from a normal adult and a male patient with paroxysmal nocturnal hemoglubinuria (PNH). Through the extensive analysis, this patient proved to be completely defective in 1F5 antigen, a newly found complement-regulatory protein, on all the blood cells tested. In normal blood cells such as lymphocytes, monocytes and granulocytes, 1F5 antigen was expressed as one of PI-anchoring proteins. In contrast to most of PNH patients, this patient reserved DAF, CD14 and CD16 at normal levels in his erythrocytes, monocytes and granulocytes. Also, there were no significant differences between the normal adult and the patient in the activities of erythrocyte acetylcholinesterase and granulocyte alkaline phosphatase which were also known to be PI-anchoring enzymes. Thus, deficiency of 1F5 antigen must be deeply related to the clinical symptoms of PNH in this patient.     

The NK-92 cell line—30 years later: its impact on natural killer cell research and treatment of cancer

The NK-92 cell line, established in 1992, mirrors all the characteristics of highly active blood natural killer (NK) cells but with much broader and greater cytotoxicity. The cell line was established from the blood cells of a patient with lymphoma and has been made widely available for research since it was deposited into the American Type Culture Collection in 1998. The worldwide distribution of NK-92 cells has led to a plethora of scientific discoveries that have greatly increased the understanding of NK-cell biology. NK-92 cells also have been developed for clinical use, overcoming the challenges of obtaining and expanding NK cells from donor or patient blood. More than 100 patients with cancer have now been treated all over the world with unmodified or genetically engineered NK-92 cells. Modified cells include high-affinity Fc-receptor expressing NK-92 cells (haNK^R) and various chimeric antigen receptor targeted haNK cells (t-haNK^TM). Infusions of either unmodified or modified NK-92 cells have been reported to be safe and efficacious, leading in some cases to disease remission even in patients who had failed multiple previous lines of therapy. It is the purpose of this review to distill the plethora of scientific data on NK-92 cells and its genetic variants, highlighting relevant experimental findings that have contributed to a better understanding of NK cell biology and summarize the therapeutic potential of these cells for treatment of cancer and infections.     

脐血间充质干细胞治疗严重性系统性红斑狼疮的临床疗效初探

目的探讨脐血间充质干细胞对严重性系统性红斑狼疮（SLE）的治疗效果。方法从健康产妇足月分娩的脐带血（UCB）中分离单个核细胞、培养制备间充质干细胞（MSCs）。MSCs培养到第三代时,取样UCB-MSCs,用流式细胞术进行性质鉴定显示：UCB-MSCs高表达CD29,CD90;不表达CD34和CD45。收集生长良好的第三代UCBMSCs。（5.0-6.0）×10^7个细胞加入100ml含1﹪人血白蛋白的生理盐水中,静脉滴注;每天1次,6-10d,一疗程;3-6个月后再用第2疗程。结果通过UCB-MSCs输注,使现有方法治疗无效的2例严重性系统性红斑狼疮患者的症状完全恢复。第1例SLE伴严重狼疮肾,狼疮脑10年,双下肢水肿,皮肤坏疽,溃疡深达骨面,剧痛难眠1年。经治医生动员她截肢保命。UCB-MSCs治疗后10d,病情明显好转,双下肢水肿几乎全部消退。治疗6个月后,11处坏疽、溃疡的疮面10个疮面基本愈合。10个月后患者的坏疽,溃疡的疮面全部愈合,能自由活动,进入正常生活。第2例SLE伴狼疮肾,狼疮脑4年,一年来贫血严重,靠每周输血维持生命。UCB-MSCs治疗二个疗程后,咳嗽,呕吐症状明显好转,浮肿和双侧胸腔积液消失,各项检查指标基本正常,不需要输血治疗。恢复休学多年的学习生活。二者均随访8年,病情稳定,没发现复发症状。结论 UCB-MSCs治疗2例严重性红斑狼疮,取得一定的疗效;这种新的技术可望成为治疗严重性系统性红斑狼疮的辅助疗法。     

Cocultivation studies with cells of patients bearing fragile X chromosomes

Summary Fourteen cocultivation studies were carried out with cells of four patients with fragile X, one obligate and two possible female heterozygotes, two female controls, and a rabbit. In all cocultivations the number of fragile X chromosomes was sharply reduced in the patient cells. The strongest effect was caused by the animal cells. A distinct difference between the two controls in the reducing ability was observed. No such difference was found between the obligate and possible heterozygotes on the one hand and the controls on the other. To test the influence of the residual serum in the mixed blood cultures, the serum of a patient's blood sample was replaced by the serum of a control. The frequency of fragile X chromosomes was not decreased by this procedure. Therefore a soluble factor is supposed to exist which is produced by normal or heterozygote cells in culture and which reduces the expression of fragile sites in patient cells.     

Circulating cancer cells in peripheral blood. A case report

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

In vitro response of cells from three patients with hairy cell leukemia to a recombinant leukocyte interferon

Mononuclear cells from the peripheral blood of two patients and from the spleen of one patient, all of whom had hairy cell leukemia, were cultured with a recombinant human leukocyte interferon (RD alpha 2-IFN). The IFN was added at concentrations of 10, 100, 1,000, and 10,000 IU/ml, and the cells were cultured for 1, 3, and 7 days. A cytocidal effect of IFN was observed only on cultured cells from the spleen at day 7. Electron-microscopic observations demonstrated that RD alpha 2-IFN induced the formation of tubuloreticular structures (TRSs) and annulate lamellae (ALs) in hairy cells, as well as in co-isolated non-leukemic cells, from all three patients. Ultrastructural examination revealed a close proximity between ALs and TRSs in co-isolated non-leukemic cells. A variability with respect to the induction of TRSs in hairy cells was observed among the three patients. In two of the three patients, the percentage of hairy cells with TRSs increased with the duration of incubation and with the dose of IFN. In the third patient, few hairy cells showed TRSs after 7 days of incubation with IFN. Our findings indicate that leukemic hairy cells may be heterogenous in their response to IFN.     

Biological characterization of human immunodeficiency virus type 1 clones derived from different organs of an AIDS patient by long-range PCR.

In order to characterize the biological properties of human immunodeficiency virus type 1 (HIV-1) variants from different tissues (peripheral blood mononuclear cells [PBMC], lymph node, spleen, brain, and lung) of one patient, we have chosen long-range PCR to amplify virtually full-length HIV proviruses and to construct replication-competent viruses by adding a patient-specific 5' long terminal repeat. To avoid selection during propagation in CD4+ target cells, we transfected 293 cells and used the supernatants from these cells as challenge viruses for tropism studies after titration on human PBMC. Despite differences in the V3 loop of the major variants found in brain and lung compared to lymphoid tissues all recombinant HIV clones obtained showed identical cell tropism and replicative kinetics. After infection of human PBMC these viruses replicated with similar kinetics, with a slow/low-titer, non-syncytium-inducing phenotype. In contrast to the prediction of macrophage tropism, drawn from the V3 loop sequence, none of these viruses infected monocyte-derived macrophages. The challenge of blood dendritic cells by these recombinant viruses in the presence of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-4 resulted in a productive infection only after adding stimulated CD4+ T lymphocytes. Therefore, the biological properties of the HIV-1 variants derived from nonlymphoid tissue of this patient did not differ from those of HIV-1 variants from lymphoid tissue with respect to tropism for primary cells such as PBMC, macrophages, and blood dendritic cells.     

Lack of transmission of deleted mtDNA from a woman with Kearns-Sayre syndrome to her child.

We have investigated the daughter of a woman with Kearns-Sayre syndrome. The woman had a high percentage of deleted mtDNA in muscle, but no deleted mtDNA was detected in fibroblasts, bone marrow, and peripheral blood cells by Southern blot analysis. With PCR, analytical sensitivity was significantly increased, and deleted mtDNA was detected in all examined tissues from this patient. The patient had healthy parents and nine healthy siblings. No deleted mtDNA was detected in blood from the mother of the patient. The patient had an uneventful pregnancy and delivered at term. Deleted mtDNA could not be detected in placenta by Southern blot analysis. With PCR, deleted mtDNA was detected in the majority of placental specimens. This finding may, however, be due to contamination with maternal DNA. The patient's daughter was healthy at age 5 mo, and morphologic examination of muscle was normal. No transmission of deleted mtDNA to the daughter could be detected by Southern blot and PCR analysis of peripheral blood cells, bone marrow, fibroblasts, and muscle. The presence of deleted mtDNA was excluded at a fractional level of less than 1:100,000 in all examined tissues from the daughter.     

Morphological and cytochemical studies of circulating Hodgkin's and Reed-Sternberg cells

Morphological and cytochemical studies of circulating neoplastic cells were carried out in a patient who presented a preterminal leukaemic phase of Hodgkin's disease (HD). Three types of abnormal cells were found in the peripheral blood: abnormal mononuclear cells, Hodgkin's cells and Reed-Sternberg cells. All neoplastic cells were cytochemically negative to Sudan black B, peroxidase and alkaline phosphatase. Some neoplastic cells were positive to PAS and all were positive to acid phosphatase, alpha-naphthylacetate esterase and beta-glucuronidase. The origin of the neoplastic population in HD is discussed.     

A monoclonal antibody detecting a 39,000 m.w. molecule that is present on B lymphocytes and chronic lymphocytic leukemia cells but is rare on acute lymphocytic leukemia blasts

Monoclonal antibody 41H.16 was produced by using the hybridoma methodology in the mouse system with cells from a patient with hairy cell leukemia as the immunogen. This antibody reacts with the majority of Slg+ cells in the peripheral blood and with all B lymphoblastoid cell lines. Reactivity with conventional Ig determinants, Fc or C3 receptors, has been excluded. The antibody reacted with cells from 68 patients with CLL but showed no reactivity with cells in 69 specimens from patients with non-T, non-B ALL. The apparent m.w. of the antigen detected by this antibody is approximately 39,000.     

Transfer of excess cholesterol from human red blood cells to plasma in vitro

This study examined the ability of plasma and plasma fractions from normolipidaemic subjects and plasma from a patient with homozygous familial high density lipoprotein deficiency (Tangier disease) to promote loss of excess cholesterol from red blood cells in vitro. Isolated high density lipoproteins were the most potent plasma fraction for removing excess cellular cholesterol. Lipoprotein-deficient plasma and human serum albumin, but not very low density lipoproteins and low density lipoproteins, also removed excess cholesterol from the red blood cells. The near absence of high density lipoproteins in plasma from the patient with Tangier disease did not result in an abnormally low rate of cholesterol loss from the enriched red blood cells. These results suggest that normal levels of high density lipoproteins are not vital for the removal of excess cholesterol from red blood cells by plasma.     

An immunohistochemical study of the distribution of ABH antigens in human submandibular glands at the light and electron microscopic levels.

The distribution of blood group antigens ABH in submandibular glands was studied at light and electron microscopy levels by applying ImmunoGold Silver Staining (IGSS) and post-embedding ImmunoGold (IGS) methods, respectively. In IGSS treated samples, a cytoplasmic and a surface form of antigen localization were discernible in the glandular parenchyma. The former was restricted to most mucous cells and to scattered serous cells: A and B antigens were demonstrated in mucous cells of A and B type glands, while H antigen appeared in most mucous and occasional serous elements regardless of the blood type of donors. The latter appeared as a strong H reactivity on cell surfaces of serous acini and ducts regardless of the patient blood type. The IGS method was applied both on non-osmicated samples embedded in LR White resin and on osmicated, Epon embedded samples. In non-osmicated tissues, antigen labelling was revealed in secretory granules and cell surfaces. Positive secretory granules were found in most mucous cells and occasional serous, intercalated, and striated duct cells. A and B antigens weakly reacted in mucous cells of A and B type glands, respectively, while strong H reactivity was seen in mucous, serous, intercalated and striated duct cells of glands of all types. Surfaces labelled with H antigen were found on both lumenal and basolateral membranes of striated ducts in glands of all types. IGS method applied on osmicated, Epon embedded samples, selectively revealed blood group antigens in secretory granules of serous cells but not in the apical vesicles of striated ductal cells. Cell surfaces were completely unreactive.     

Optimization of an enrichment process for circulating tumor cells from the blood of head and neck cancer patients through depletion of normal cells

The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 × 10⁹ to 8.04 × 10³ cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patient's blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log₁₀) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominately epithelial or mesenchymal characteristics?). Biotechnol. Bioeng. 2009;102: 521–534. © 2008 Wiley Periodicals, Inc.     

Stimulation of lymphocytes from subacute sclerosing panencephalitis patients by defined measles virus antigens

The lymphoproliferative response of human peripheral blood mononuclear cells to different measles virus antigen preparations was studied with lymphocytes from 38 measlesseropositive healthy donors and 4 subacute sclerosing panencephalitis patients. The response was very weak or absent in all of the controls and in three of the subacute sclerosing panencephalitis patients. The fourth subacute sclerosing panencephalitis patient had fluctuating levels of lymphocyte stimulation by measles antigens. The response was very strong for several months and during this time the parameters of the test system were characterized. It was discovered that a membrane preparation of measles-infected cells caused stimulation equal to that of highly purified virions. Purified measles ribonucleoprotein also induced specific stimulation, although lower than that seen with other types of measles antigens. Results of experiments on stimulation kinetics and antigen dose responses were compatible with antigen-specific stimulation. Enriched T cells were more vigorously stimulated than unfractionated peripheral blood mononuclear cells suggesting that this transformation test is specific for T cells.     

Alterations of glycosphingolipid-based blood group antigen expression on erythrocytes and in plasma studied on consecutive samples after a blood group O to A bone marrow transplantation.

A blood group A1Le(a-b+) individual with chronic myeloid leukaemia had received a bone marrow graft from an HLA-identical OLe(a+b-) donor. Twelve months after bone marrow transplantation (BMT), the red blood cells of the patient became agglutinable with anti-A blood group reagents. To elucidate whether the blood group A antigen expression was of plasma or of bone marrow origin, total non-acid glycosphingolipid fractions were prepared from red blood cells and plasma collected 17 months after BMT, and from plasma collected 13, 15 and 19 weeks after BMT. The glycolipid fractions were analysed by thin-layer chromatography and immunostained with monoclonal A-antibodies, and permethylated and permethylated-reduced derivatives of selected plasma samples were analysed by mass spectrometry. The results strongly indicate the presence of host bone marrow-produced blood group A red blood cells. Furthermore, the presence of a blood group H active pentaglycosylceramide type 1 (H-5-1) (Table I), characteristic for an OLe(a-b-) secretor, was seen in plasma 3-4 weeks before clinical chronic graft versus host disease (GVHD). After treatment of chronic GVHD, this expression disappeared. The blood group ALeb (A-7-1) antigen produced by the recipient seems to be present and to increase with time in all plasma samples. This also seems to be the case for the Leb and A-6-1 antigens.     

Vaccination of a melanoma patient with mature dendritic cells pulsed with MAGE-3 peptides triggers the activity of nonvaccine anti-tumor cells

We previously characterized the CTL response of a melanoma patient who experienced tumor regression following vaccination with an ALVAC virus coding for a MAGE-A3 Ag. Whereas anti-vaccine CTL were rare in the blood and inside metastases of this patient, anti-tumor CTL recognizing other tumor Ags, mainly MAGE-C2, were 100 times more frequent in the blood and considerably enriched in metastases following vaccination. In this study we report the analysis of the CTL response of a second melanoma patient who showed a mixed tumor response after vaccination with dendritic cells pulsed with two MAGE-A3 antigenic peptides presented, respectively, by HLA-A1 and HLA-DP4. Anti-MAGE-3.A1 CD8 and anti-MAGE-3.DP4 CD4 T cells became detectable in the blood after vaccination at a frequency of approximately 10(-5) among the CD8 or CD4 T cells, respectively, and they were slightly enriched in slowly progressing metastases. Additional anti-tumor CTL were present in the blood at a frequency of 2x10(-4) among the CD8 T cells and, among these, an anti-MAGE-C2 CTL clone was detected only following vaccination and was enriched by >1,000-fold in metastases relative to the blood. The striking similarity of these results with our previous observations further supports the hypothesis that the induction of a few anti-vaccine T cells may prime or restimulate additional anti-tumor T cell clones that are mainly responsible for the tumor regression.     

Idiotypic analysis of a B cell clone with anti-intermediate filament specificity in a patient with Sj?gren's syndrome: involvement of five subpopulations producing different immunoglobulin isotypes

An IgM paraprotein from patient LP with Sj?gren's syndrome exhibited an antibody activity to intermediate filaments (IMF) of cells from all vertebrates examined, and appeared to recognize several classes of IMF (i.e., vimentin, desmin, and keratin). A mouse monoclonal anti-idiotype (Id) antibody, K4A, was prepared against the IgMk (LP) and used as a specific probe in two-color immunofluorescence to examine the extent of clonal involvement in the patient's blood and bone marrow mononuclear cells (MNC). Twenty to 30% of MNC in her blood samples were IgMk+ plasmablasts with morphologic similarity to Waldenstr?m's macroglobulinemia cells. IgG+ and IgA+ plasmablasts were demonstrated in lower frequencies (approximately 2%). Almost all of the IgM+ cells and approximately 80% of the IgG+ cells and IgA+ cells in the blood were reactive with the K4A anti-Id antibody. Immunoglobulin (Ig) subclass analysis revealed that the K4A Id was expressed by IgG1+, IgG3+, IgA1+ and IgA2+ plasmablasts. Similar observations were obtained with bone marrow samples, although the proportion of Id+ cells among IgG+ or IgA+ cells was lower in marrow than in blood. IgG and IgA fractions isolated from the patient's serum were also shown to contain anti-IMF activity. Ig biosynthetic analysis of blood MNC revealed that the K4A anti-Id antibody precipitated not only IgM but also IgG and IgA. Because cells simultaneously producing two different Ig isotypes were not detected, these results indicate the presence of five separate subpopulations of the K4A Id+ neoplastic clone. The data thus suggest the occurrence of a neoplastic or pre-neoplastic transformation event before the switching of Ig heavy chain isotypes, and imply a role for the IMF antigen in the exaggerated proliferation and differentiation along five of the nine potential intraclonal pathways.     

Immunocytodiagnosis of carcinocythemia in disseminated breast cancer

A small number of large, malignant mononuclear cells were found in the blood of a patient with disseminated breast carcinoma. The detection of these scarce cells was facilitated by examining leukocyte concentrates. Cytochemical and immunochemical studies showed that these were epithelial cells, not hemopoietic cells. These findings, when correlated with the clinical features and the cytologic findings in blood and marrow, suggested that the malignant mononuclear cells in the blood were breast carcinoma cells. This study demonstrates the technical feasibility of accurately identifying cells in blood, which may stimulate interest in the study of cancer cells in the circulating blood. The method may also be applied to other cytologic specimens, such as fine needle aspirates, that contain only a few neoplastic cells.     

Modified Leukocyte Filter Removes Tumor Cells from the Salvaged Blood

Background

Intraoperative blood salvage, an effective blood conservation strategy, has not been applied in onco-surgery, because of potential malignant cell contamination. In this study we tested effectiveness of a modified leukocyte depletion filter (M-LDF) for removal of tumor cells.

Materials and Methods

The effects of M-LDF and regular LDF on removal of cells (HepG2 cell line) were compared. The safety of M-LDF was tested with blood (collected and washed during onco-surgery), the salvaged blood mixed with tumor cells from the solid tumor of the same patient, or mixed with HepG2 cells (n=30 in each protocol). Cancer cells were identified by flow cytometry, culture and bioassay with and without filtration.

Results

M-LDF removed 5-log of HepG2 and nucleated cells, which was much higher than regular LDF, and cells were destroyed when they passed through M-LDF. Cytokeratin-positive cells in all samples were removed by M-LDF. Invasive growth adherent cells were found in most of unfiltered samples and 67% of the inoculated nude mice developed tumors in LDF-treated sample. Neither adherent cells nor nude mice developed tumors were found in M-LDF-treated samples.

Discussion and Conclusion

Since M-LDF can effectively remove and destroy cancer cells in the salvaged blood, it has great potential for clinical application.