Lymphocyte depletion during treatment with intensive chemotherapy for cancer

Recently we have observed an increased incidence of opportunistic infections in patients treated with intensive chemotherapy for cancer. Because T-cell depletion is associated with similar clinical events in human immunodeficiency virus infection and after bone marrow transplantation, we have analyzed peripheral blood lymphocyte populations in a series of patients during treatment with intensive chemotherapy for cancer. Although neutrophil, monocyte, and platelet numbers consistently recovered to greater than 50% of pretreatment values after each sequential cycle of therapy, lymphocyte numbers did not recover within the same time period. B cells decreased rapidly from a mean value of 149 +/- 46/mm3 before chemotherapy to 4 +/- 1/mm3 during chemotherapy (P = .01). CD4+ T cells decreased from a mean of 588 +/- 76/mm3 before chemotherapy to 105 +/- 28/mm3 during chemotherapy (P = .0002) and CD8+ T cells decreased from a mean of 382 +/- 41/mm3 before chemotherapy to 150 +/- 46/mm3 during chemotherapy (P = .0009). Natural killer cell numbers did not show significant declines (171 +/- 30/mm3 before, 114 +/- 24/mm3 during, P = .19). Based on the history of opportunistic complications in patients with other disorders who display similar degrees of CD4+ T-cell lymphopenia and preliminary observations in this population, immune incompetence could surface as a dose-limiting toxicity for highly dose-intensive chemotherapy regimens.     

Incidental Non-Inguinals Hernias in Totally Extra-Peritoneal Hernia Repair

Introduction

Totally extra-peritoneal (TEP) inguinal hernia repair allows identification and repair of incidental non-inguinal groin hernias. We assessed the prevalence of incidental hernias during TEP inguinal hernia repair and identified the risk factors for incidental hernias.

Materials and Methods

Consecutive patients undergoing TEP repair from May 2005 to November 2012 were the study cohort. Inspection for ipsilateral femoral, obturator and rarer varieties of hernia was undertaken during TEP repair. Patient characteristics and operative findings were recorded on a prospectively collected database.

Results

A total of 1,532 TEP repairs were undertaken in 1,196 patients. Ninety-three patients were excluded due to incomplete data, leaving 1,103 patients and 1,404 hernias for analyses (1,380 male; 802 unilateral and 301 bilateral repairs; median age, 59 years). Among the 37 incidental hernias identified (2.6% of cases), the most common type of incidental hernia was femoral (n=32, 2.3%) followed by obturator (n=2, 0.1%). Increasing age was associated with an increased risk of incidental hernia, with a significant linear trend (p<0.01). The risk for patients >60 years of age was 4.0% vs 1.4% for those aged <60 years (p<0.01). Incidental hernias were found in 29.2% of females vs 2.2% of males, (p<0.0001). Risk of incidental hernia in those with a recurrent inguinal hernia was 3.0% vs 2.6% for primary repair (p=0.79).

Conclusions

Incidental hernias during TEP inguinal hernia repair were found in 2.6% of cases and, though infrequent, could cause complications if left untreated. The risk of incidental hernia increased with age and was significantly higher in patients aged >60 years and in females.     

Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures

There is a growing consensus that clinical practice in the areas of bone marrow (BM) transplantation and gene therapy will rely on the ex vivo expansion of hematopoietic cells. We report here on the development of continuously perfused culture systems (bioreactor systems) that expand human stem and progenitor cells from BM mononuclear cell (MNC) populations obtained without cell enrichment. In three separate experiments, 10 bioreactors were each inoculated with 3 x 10(7) BM MNC from patients undergoing marrow harvest for autologous transplantation. At various times thereafter (between days 6 and 16), duplicate bioreactors were harvested and cells were analyzed. The bioreactors contained three cell populations that were analyzed separately: nonadherent cells; cells that were loosely adherent to the endogenously formed stromal layer; and an adherent cell layer that required trypsinization for removal. Total cell numbers increased continuously to give an overall 10-fold (range, 8- to 11-fold) expansion by day 14. The adherent stromal layer significantly expanded to more than 2 x 10(7) cells, but remained less than 6% of the total cell population. Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this expansion was greater than that for total cells, CFU-GM were enriched by as much as fourfold by day 14. Burst-forming unit-erythroid (BFU-E) numbers peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to 18-fold) expansion obtained on day 8. In contrast to CFU- GM, which were predominantly nonadherent, BFU-E were more evenly distributed between the three cell populations. Stem cell activity was measured by the long-term culture-initiating cell (LTC-IC) limiting dilution assay. The number of LTC-IC per reactor consistently increased with time in all cultures, resulting in a 7.5-fold (range, 3.4- to 9.8- fold) expansion. In summary, more than 3 billion cells, containing 12 million CFU-GM, were reproducibly generated from the equivalent of a 10 to 15 ml BM aspirate. These data indicate that small numbers of BM MNC can be readily expanded ex vivo in continuous perfusion cultures, and that such ex vivo expansion may have direct applications in clinical and experimental BM transplantation.     

Identification and characterization of plasma cells in normal human bone marrow by high-resolution flow cytometry

The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma cells were located in a unique position in the correlation of forward light scattering, orthogonal light scattering, and immunofluorescent-labeled CD38. The identity of the sorted cell populations was confirmed by microscopic examination of Wright's stained slides and slides stained for cytoplasmic immunoglobulin using polyclonal antibodies reactive with light chains; ie, anti-kappa fluorescein isothiocyanate and anti lambda phycoerythrin (PE). The purity of the sorted plasma cells was greater than 97% (n = 4). The average frequency of plasma cells in normal bone marrow aspirates was low--0.25% of the nucleated cells (n = 7)--but surprisingly consistent between individuals (SD = .05; range 0.14% to 0.30%). A detailed analysis showed two distinct populations of plasma cells: (1) A population relatively smaller by forward light scattering expressed CD22, CD35, and sigE and was identified as early plasma cells (ie, lymphoplasmacytoid), and (2) a population larger by forward light scattering lacked these markers and was identified as mature plasma cells. The antigenic profile of the normal plasma cells was determined in two-color immunofluorescence studies. The expression of cell surface immunoglobulin G (IgG), IgA, IgE, IgD, IgM, and the cell surface antigens CD10, CD11b, CD13, CD11c, CD14, CD15, CD16, CD19, CD22, CD20, CD33, CD35, CD45, and HLA-DR was determined on the plasma cells. A significant heterogeneity in cell surface antigen expression was observed within the plasma cell population. Unexpectedly, myeloid- specific cell surface antigens such as CD33 and CD13 and the early B- cell antigen identified by CD10 were expressed on a proportion of plasma cells. These observations imply that the association of myeloid and early B-cell markers described in multiple myeloma may not be associated with the neoplasia but is a normal phenomenon.     

cis-Retinoic acid stimulates the clonal growth of some myeloid leukemia cells in vitro

We studied the effects of cis-retinoic acid (cisRA) on the clonogenic growth of samples of leukemic cells from 35 patients with acute nonlymphocytic leukemia (ANLL). We observed significant inhibition of leukemic colony growth in 17 samples by 10(-7) to 10(-6)M cisRA. However, we found that retinoid exposure resulted in striking stimulation of clonal growth in ten samples at the same drug concentrations. With the exception of cases with promyelocytic features, there was no morphologic or functional evidence that cisRA induced the leukemic blasts to differentiate. Both inhibition and stimulation were dose-dependent and observable at pharmacologically achievable levels of cisRA. Leukemic cells with monocytic features more frequently demonstrated a stimulatory response than did those without monocytic features. Depletion of T lymphocytes and monocytes did not alter the type of growth response. Assays for cellular retinoic acid- binding protein (CRABP) were performed on five samples (two with inhibitory growth responses, two with stimulatory responses, and one with no growth) and failed to reveal detectable levels of CRABP in any case. The addition of cisRA to liquid suspensions of leukemic cells produced no significant change in the number of viable cells. We conclude that the effects of cisRA on leukemic colony growth are not cytotoxic and not mediated by T lymphocytes, monocytes, or CRABP. More importantly, cisRA appears to enhance the growth of certain human leukemia cells in vitro. Taking into account the increasing use of retinoids in clinical trials for patients with leukemia, the latter findings may represent a significant cautionary note.     

Diethylstilbestrol selectively modulates plasma coagulation protein synthesis by the perfused rat liver

The combined effects of orchiectomy and estrogen administration on synthesis of selected hepatic secretory proteins--antithrombin (AT) III, plasminogen, fibrinogen, factor II (prothrombin), factor VII, fibronectin, and albumin--were studied using the isolated rat liver perfused in vitro for ten hours. Male rat liver donors underwent orchiectomy under ether anesthesia and then received 5.0 mg of diethylstilbestrol (DES) by subcutaneous pellet implantation or a placebo pellet; 14 days later the livers were extracted and perfused in vitro for ten hours. In DES experiments, 1.0 mg of DES was also added directly to the liver perfusate at the outset. Pretreatment with DES resulted in significant increases in cumulative synthesis of factors II (65%) and VII (76%) and significant reduction in cumulative synthesis of both antithrombin III (20%; P = .03) and plasminogen (27%; P less than .01) compared to control perfusions, but synthesis of fibrinogen, fibronectin, and albumin was not significantly affected by addition of the hormone. Plasma samples collected from rat liver donors that had received DES showed similar effects on protein concentrations: significant decreases in concentration of plasminogen and antithrombin III were apparent with no significant changes in concentrations of fibrinogen, fibronectin, or albumin. In additional perfusions, "dose- response" experiments were conducted in which rat liver donors received a subcutaneous DES pellet of 0.5, 5.0, or 50 mg. Synthesis of plasminogen in this group of perfusions was progressively decreased as the concentration of DES administered to the rat liver donor increased. Synthesis of AT III was reduced to the same degree by 5.0 or 50 mg of DES, both being substantially lower than the 0.5-mg experiments. Concentrations of these two proteins in plasma samples from rat liver donors showed changes quite similar to those seen in perfusion experiments; however, plasma fibrinogen concentrations were not different among the three groups of rats.     

B-cell homotypic adhesion through exon-A restricted epitopes of CD45 involves LFA-1/ICAM-1, ICAM-3 interactions, and induces coclustering of CD45 and LFA-1

Lymphocyte interactions with other leukocytes and other cell types, as well as with components of the extracellular matrix, are one of the key steps in the immune response. Three novel monoclonal antibodies (MoAbs) have been produced and selected for their ability to induce intercellular adhesion in B cells. These three MoAbs immunoprecipitated a polypeptide of 220 kD, displaying specific phosphotyrosine phosphatase activity that has been identified as CD45. These MoAbs recognize epitopes located on the alternative spliced exon-A-encoded region of CD45. These epitopes are of polypeptidic nature, but they can be masked by addition of carbohydrate during CD45 biosynthesis. Interestingly enough, CD45 epitopes recognized by these MoAbs appeared to be selectively expressed on both peripheral blood and tonsillar B lymphocytes as well as on peripheral blood natural killer (NK) cells. CD45-mediated intercellular adhesion was abrogated upon incubation with anti-leukocyte function-associated antigen 1 (anti-LFA-1), intercellular cell adhesion molecule 1 (ICAM-1), and ICAM-3 MoAbs, thus indicating that this phenomenon involved both LFA-1/ICAM-1 and LFA- 1/ICAM-3 cell adhesion pathways. Moreover, CD45-mediated cell aggregation was also inhibited by preincubation with some conventional anti-CD45 MoAbs. Interestingly, the triggering of cell aggregation through CD45 induced membrane surface relocation of CD45 and LFA-1 molecules, with both of them colocalizing at cell-cell contact areas of B-cell aggregates. Studies with inhibitors of both phosphotyrosine phosphatase and tyrosine kinase activities suggest that CD45 phosphotyrosine phosphatase activity could be involved in CD45-mediated cell aggregation. Taken together, these results support the notion that CD45 is a key molecule in the regulation of LFA-1-mediated cell-cell interactions.     

New variant of von Willebrand disease type II with markedly increased levels of von Willebrand factor antigen and dominant mode of inheritance: von Willebrand disease type IIC Miami

A variant of von Willebrand disease (vWD) was identified in six members of a kindred spanning four generations. The proband was a 46-year-old woman with a lifelong history of bleeding, a prolonged bleeding time (> 15 minutes), markedly elevated von Willebrand factor (vWF) antigen (vWF:Ag = 2.09 U/mL), slightly reduced ristocetin cofactor activity, and a plasma vWF multimer pattern similar to that of vWD type IIC. Similar findings were observed in her three children, mother, and brother. In affected family members, platelet and plasma vWF multimer patterns were discrepant with higher molecular weight multimers observed in platelet vWF. Following a 1-Des-amino-8-D-arginine vasopressin (DDAVP) challenge, the proband failed to normalize her bleeding time even though vWF: Ag rose by 70% and higher molecular weight multimers were increased slightly. Genetic studies were consistent with autosomal dominant inheritance of a mutation within the vWF gene. By sequencing of cloned genomic DNA, mutations were excluded in exons 4, 5, 14, and 15, which encode regions of the vWF propeptide proposed to be important in multimer biosynthesis. Mutations also were excluded in exons 28 to 31, which encompass the known mutations that cause vWD types IIA, IIB, and B. This new variant of vWD, characterized by autosomal dominant inheritance, a qualitative defect that resembles vWD type IIC, and increased plasma vWF:Ag, was tentatively designated vWD type IIC Miami.     

In situ immunologic characterization of cellular constituents in lymph nodes and spleens involved by Hodgkin's disease

The cellular constituents in lymph nodes and spleens of patients with Hodgkin's disease were studied with a series of monoclonal antibodies directed against human thymocyte, peripheral T-cell, and la antigens. Utilizing both an immunoperoxidase technique on frozen tissue sections and indirect immunofluorescence on cell suspensions, wer found that a majority of lymphocytes were T cells, since they stained with anti-T1 and anti-T3 antibodies, which react with all peripheral T cells. In addition, most of these cells were reactive with anti-T4 antibody, which defines the helper/inducer T-cell population, whereas only a minority of cells stained with anti-T5 and anti-T8 antibodies, which are reactive with suppressor/cytotoxic T cells. Moreover, a large proportion of T cells expressed T10 antigen, which is found on activated T cells. A minority of the T cells also expressed la antigen(s), again suggesting that some of the T cells are activated. In contrast, the Reed-sternberg cells did not react with any of these anti- T-cell antibodies or with anti-IgM antiserum, but displayed strong membrane and cytoplasmic staining with anti-la antibody. Taken together, these findings suggest that Reed-Sternberg cells are not of T- cell lineage but may be derived from antigen-presenting reticulum cells in the thymus-dependent areas of lymphoid tissues; these cells are normally associated with T4+ cells.