Racial Differences in Accepting Pegfilgrastim Onpro Kit (On-Body Injector) Use Among Cancer Patients.

Background: 1.1.Neulasta Onpro kit eliminates need for additional clinic visit after chemotherapy. Given the racially diverse population in our institution, we investigated acceptance of Onpro kit among patients on chemotherapy. Research Design and Methods: 1.2.Single-institution, retrospective review conducted in patients with GI tumors who received Onpro kit within 1 hour of completion of systemic chemotherapy from Jan 2014 through Jan 2018. Clinic/nursing notes and pharmacy records were reviewed to identify patients who refused Onpro kit and to discern reasons for refusal, including racial reason. Results: 1.3.Total 238 orders for kit were voided amongst 68 patients (Caucasian 41; African American 7; Spanish 3; Asian 17). Overall, 15/68 patients refused kit (22%) of these 87% were Asian. The reasons for refusal included dislike of bulky attachment to skin, request to place kit on stomach instead of arm, trepidation over unwitnessed administration of drug, fear of reaction, disposal at home, fear of pain, lack of confirmation of proper dose administration, and need for MRI. Conclusions: 1.4.While Onpro kit is an attractive alternative, 22% of patients with voided orders, mainly of Asian race, declined its application. We believe the current study represents the first look at important racial differences in accepting Onpro kit. Consideration of patients' cultural heritage, race, ethnicity and education may facilitate communication between physicians and patients to achieve optimal cancer care.

A novel reduced intensity regimen for allogeneic hematopoietic stem cell transplantation associated with a reduced incidence of graft-versus-host disease.

SUMMARY: In all, 55 patients at high risk or ineligible for a conventional allogeneic hematopoietic stem cell transplant (HSCT) received a regimen consisting of extracorporeal photopheresis, pentostatin, and reduced dose total body irradiation. The median age was 49 years (18-70 years); 44 received a sibling and 11 an unrelated HSCT; 44% were over the age of 50 years and 31% had undergone a prior HSCT. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate. Full donor chimerism was documented in 98% by day +100. The 1000-day nonrelapse mortality was 11%. The median follow-up is 502 days (154-1104 days). The 1- and 2-year overall survival (OS) and event-free survival (EFS) are 67, 58 and 55%, and 47%, respectively. Patients who had not received a prior HSCT or had less than three prior chemotherapy regimens had a 71% OS and 67% EFS at 1 year. Greater than grade II aGVHD developed in 9% and chronic GVHD (cGVHD) in 43%, and extensive in 12% and limited in 31%. Of the patients, 86% who engrafted had a disease response, 72% had complete and 14% partial responses. This novel reduced intensity preparative regimen was well tolerated and associated with a low incidence of transplant-related mortality and serious acute and cGVHD.

MHC class I antigens regulate CD3-induced tyrosine phosphorylation of proteins in T cells.

The MHC class I antigens enhance the T cell response to various mitogenic stimuli. Class I antigens co-cap and associate with the CD3 structure on these cells. The present work shows that co-aggregation of these MHC antigens with CD3 induces a sustained elevated Ca2+ response in T cells. The duration required for a 50% decline in peak response is five to 10 times longer when class I antigens are cross-linked with CD3 as compared to that when cells are stimulated through CD3 alone. Further analysis reveals that class I antigens prolong the duration of CD3-induced tyrosine phosphorylation of several proteins. No protein tyrosine kinase activity is found associated with these MHC antigens which could explain their influence on CD3 activation. Similarly, class I antigens do not depend on the membrane-bound protein tyrosine phosphatase CD45, since they elevate the degree of CD3-induced Ca2+ response in a CD45-deficient Jurkat cell line. On the contrary, in a CD45- HPB cell line defective in CD3 signaling, co-aggregation of class I antigens with CD3 does not induce Ca2+ flux. Therefore, the effect of class I antigens on CD3 function depends on the ability of CD3 to transduce a signal. Furthermore, cytofluorometric studies show that cross-linking of class I antigens with CD3 inhibits the internalization of the latter. Thus, class I antigens seem to prolong the duration of signal transduction through CD3 by retarding its down-regulation.

Defective signal transduction via T-cell receptor-CD3 structure in T cells from rheumatoid arthritis patients.

T cells from patients with active RA are known to produce low levels of IL-2 and proliferate poorly in response to various mitogenic stimuli. The present work shows that cross-linking of CD3 antigen on patients' T-cell surface induces two- to threefold lower Ca2+ response than in T cells from age-matched controls. Immunofluorescence studies indicate that the attenuated signal may be due to the suppressed expression of CD3 and/or CD45 molecules on patients' T cells. In the majority of the patients, the level of CD45 expression is reduced by 60%-70% as compared with that in the control T cells. Therefore, the poor mitogenic response of patient cells is apparently due to a defect in early stages of signal transduction through the T-cell receptor (TCR-CD3).

Phospholipase C-gamma 1 association with CD3 structure in T cells.

Recently, we and others have reported tyrosine phosphorylation of phospholipase C-gamma 1 (PLC gamma 1) enzyme after CD3 activation of T cells, and have proposed that PLC gamma 1 mediates signal transduction through the T cell receptor (TCR/CD3). Here, using immunoblotting and immune complex PLC assays, we show that CD3 stimulation of Jurkat cells induces the association of PLC gamma 1 enzyme with CD3 complex. PLC activity is also found to co-precipitate with the CD3 zeta chain from activated cells. In addition, in vitro PLC assays show that CD3 activation leads to about 10-fold stimulation of PLC gamma 1 activity. These results, along with the observation that Jurkat cells preferentially express PLC gamma 1, indicate that PLC gamma 1 participates in CD3 signaling.

PLC gamma 1, a possible mediator of T cell receptor function.

Stimulation of T cell antigen receptor (TCR/CD3) following the recognition of peptide-major histocompatibility antigen complex induces phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, the phospholipase C (PLC) enzyme mediating this process has not been identified. We report that PLC gamma 1 protein is expressed in human T cells. It is a phosphoprotein, and the activation of cyclic AMP-dependent protein kinase (PKA) or of protein kinase C (PKC) with forskolin or phorbol ester, respectively, increases the level of phosphorylation. CD3 stimulation of T cells induces tyrosine phosphorylation of PLC gamma 1 and causes 8-10-fold higher yield of PLC activity with anti-phosphotyrosine antibody (APTyr Ab) from activated cells than from non-activated cells. Genistein, an inhibitor of protein tyrosine kinase, decreases this yield of AP-Tyr Ab-bound PLC activity from activated cells and lowers the level of Ca2+ mobilization. Furthermore, phorbol ester and forskolin treatment of cells before CD3 stimulation reduces the level of tyrosine phosphorylation of PLC gamma 1 and the PLC activity associated with APTyr Ab. These results suggest that CD3 stimulation activates PIP2 hydrolysis by inducing tyrosine phosphorylation of PLC gamma 1, which is regulated negatively by PKC and PKA.

Involvement of major histocompatibility complex class I antigens in T cell activation.

During the last few years ample evidence has been collected indicating a regulatory role for major histocompatibility complex class I antigens (Ag) in T cell activation. However, due to differential effects (stimulatory and inhibitory) of anti-class I antibodies (Ab) observed under different conditions, no coherent scheme of the mechanism of action of these Ag has emerged. Here, we present evidence that the mode of action of anti-class I Ab depends upon the presence or absence of monocytes/macrophages (M phi) in the culture. The Ab inhibit Ag presentation by binding to M phi. Coating of tetanus toxin -pulsed M phi with anti-class I Ab is sufficient to suppress T cell activation. On the contrary, these Ab enhance lectin- as well as phorbol ester-induced T cell activation in the absence of M phi. Cross-linking of class I Ag on T cell surface mobilizes cytoplasmic Ca2+, and also enhances the CD3-induced Ca2+ flux inside the cells indicating a functional relationship between CD3 and class I Ag. Though surface modulation and immunoprecipitation experiments do not indicate any physical association between these two types of molecules on the T cell surface, capping studies show that cross-linking of class I Ag induces an association of these Ag with CD3. Binding of anti-CD3 Ab enhances the strength of association between CD3 and class I Ag, and the former co-caps completely with the latter. Based on these observations we propose that during antigen presentation M phi (or Ag-presenting cells) and T cells, besides interacting via peptide--class II Ag/CD3--T cell receptor complex formation, also interact through class I Ag. The latter interaction may stabilize the contact formation between T cells and Ag-presenting cell and support T cell activation.

Monoclonal antibody to human cross-linked fibrin.

Cross-linked Fibrin II was prepared using Kabi grade (L) fibrinogen. Fibrin plasmic digest was separated on Sepharose CL-6B. Fragments Mr 135-300 kDa were used to immunize 6-9 weeks old female BALB-c mice. A stable hybridoma secreting monoclonal antibody (MAb) TD-1 (IgG 2a, Kappa) was prepared by fusion using myeloma cells (P3-NS1/1-Ag4-1) and immunized cells. Fibrinogen and plasmin digest of fibrinogen in serial dilutions did not compete with the immunizing antigen. To prove that TD-1 binds specifically to cross-linked fibrin, immunoprecipitation with S. aureus and affinity chromatography were performed. In both experiments, we demonstrated that TD-1 binds specifically to a protein Mr greater than 200 kDa which is found in XL-fibrin and not fibrinogen. Reduced samples showed the antibody bands (heavy and light chains) and three protein bands, Mr greater than 80 kDa (gamma-gamma dimer), Mr greater than 45 kDa (beta chain of fragment D) and Mr greater than 16 kDa (alpha chain from fragment D) were present. TD-1 reacted strongly with HPLC fraction of the immunizing antigen Mr 220 kDa (probably DD/E complex). Affinity binding constants (Scatchard Plot Analysis) were determined. The highest affinity was obtained with XL-fibrin fraction Mr 220 kDa, KD = 1.39 X 10(-8) and high molecular weight XL-fibrin fragments, KD = 1.6 X 10(-7). Fragment DD had KD of 2.8 X 10(-6). These results suggest that TD-1 is specific for the DD region of human cross-linked Fibrin II.

Two variants of DRw52, DR3, and DQw2 specificities distinguish two different DR3-bearing extended haplotypes.

HLA-DR beta and DQ beta MHC antigens present on B-lymphoblastoid cell lines homozygous for either [HLA-B8,DR3,SC01] or [HLA-B18,DR3,F1C30] were studied by two-dimensional gel electrophoresis. Comparison of neuraminidase-treated DR proteins from these cells showed that DR3-bearing cells express two DR beta genes. However, one DR beta chain (beta a) is nonpolymorphic, whereas the other beta chain (beta b) is polymorphic. Two variants of DR beta (DR3 or DRw52) and two of DQ beta (DQw2) were found, with all the examples of each extended haplotype carrying only one of these sets of variants. The variants thus absolutely distinguished the two DR3-bearing extended haplotypes. These results support the hypothesis of extended haplotypes at the protein level, and demonstrate the fixity of alleles on them in the HLA-B-D/DR region.

Expression of extra class I major histocompatibility antigens on T-cell acute lymphoblastic leukemia (ALL) lymphoblasts.

The presence of extra HLA antigens has been demonstrated, serologically and biochemically, on the surface of lymphoblasts from a patient with acute lymphoblastic leukemia of the T-cell subtype (T-ALL). Family analysis of this patient revealed the presence of the expected antigens, plus an additional HLA antigen (A24) which could be demonstrated by cytotoxicity on the lymphoblasts. Absorption studies revealed that the lymphoblasts had the ability to remove cytotoxic antibodies from alloantisera; similarly, absorption of these alloantisera with normal cells removed the reaction against the extra antigen from the lymphoblasts. The extra HLA molecules were also demonstrated by one-dimensional IEF. Two heavy chain-like molecules, together with the beta 2m subunit, were obtained after removal of appropriate antigens from externally labeled leukemia cells by the use of monoclonal antibody W6/32, which detects a class I specific determinant. The pI of the one heavy chain was shown to be very similar to that of the serologically detected A24. Our data thus suggest that the extra antigens detected by serological reagents may have been due to activation of silent class I MHC gene(s) at the protein level.

IEF patterns of HLA-B13 antigens from Orientals and Caucasians.

HLA-B13 antigens were isolated from metabolically labeled cell extracts from Caucasian and Oriental donors by means of an HLA-B13-specific monoclonal antibody, SY1. Ethnic differences in B13 molecules were identified by one-dimensional isoelectric focusing in which the pI of desialated Oriental B13 molecules was found to be higher than that of Caucasians. An additional Caucasian variant pattern was detected by peptide mapping using limited proteolysis in sodium dodecyl sulfate analyzed by polyacrylamide gel electrophoresis. Dual allotypic determinants for B13 molecules were recognized by two HLA-B13-specific monoclonal antibodies, SY1 and Tu110, as determined by their sensitivity to complement-dependent cell lysis. Whereas the SY1 target epitope was shared by both ethnic B13 molecules, the two ethnic B13 molecules carried different Tu110 target structures. The Caucasian variant molecules appear to carry altered allotypic determinants which are recognized by both SY1 and Tu110 antibodies. This study suggests that the HLA-B13 private structure may comprise two epitopes recognized by SY1 and Tü110 antibodies, respectively, whose binding sites overlap. Present data also suggest that the private determinant was already present when the two racial groups diverged, and thus the mutations which gave rise to the variants may be of relatively recent origin.