Low Secretors Research Articles

Improved cell line development by a high throughput affinity capture surface display technique to select for high secretors

A novel process is described which permits rapid and objective selection of rare cells from a heterogeneous population based on quantity of secreted target protein. The process involves construction of an immobilised affinity surface display matrix that specifically binds secreted target product which is then detected using a fluorescent labelled ligand. Cells with the highest fluorescence can then be sorted using conventional flow cytometric technology. Overall, the whole process can be completed in less than 4 h during which time in the region of five million cells can be analysed. Cells are rapidly selected for in a quantitative manner compared to traditional methods which can take several months and have a reduced probability of finding low abundance high secretors due to practical limitations imposed on the number of cells which can be screened.

Soluble HLA-I in rheumatic diseases

Objective: To study serum levels of Class I soluble HLA (sHLA-I) in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis or dermatomyositis (PM/DM) or scleroderma and to assess the possible influence of ethnic factors on concentration in each disease group. Methods: Solid-phase enzyme linked immunoassay was used to measure sHLA-I in the serum of 385 patients with varied ethnic backgrounds (American-Caucasians, African-Americans, Georgian-Caucasians) with rheumatic diseases. Studies on patients were compared to similar measurements of 189 healthy individuals. Results: Mean sHLA-I levels were significantly higher in patients with SLE than those observed in healthy individuals or other rheumatic diseases. Highest concentrations were present in Georgian-Caucasian patients with SLE. American-Caucasian patients with RA or scleroderma had higher sHLA-I levels than normal Caucasian individuals. The majority of patients with PM/DM in all ethnic subgroups were low secretors of sHLA-I. Conclusion: Mechanisms underlying the secretion of sHLA-I appear to differ among the rheumatic diseases studied and various ethnic groups. These genetic differences in sHLA-I secretion could be associated with ethnic and pathophysiologic differences among these rheumatic diseases.

Nitric oxide may trigger lactation in humans

Objective:To determine whether nitric oxide is synthesized in the breast and plays a role in lactation. Design:Concentrations of biopterin, neopterin, and the total concentration of nitrite plus nitrate, a marker for nitric oxide generation were measured in 242 samples of breast milk obtained from 39 women during postpartum days 1 to 30. The total concentration of nitrite plus nitrate was measured in 17 sets of breast milk and serum obtained from 17 women on postpartum day 4 or 5. Results:(1) The total concentration of nitrite plus nitrate rose and peaked just before an increase in the volume of milk secreted was observed. (2) The total concentration of nitrite plus nitrate in breast milk was not correlated with that in the serum. (3) High levels of neopterin and biopterin were found in breast milk. (4) The volume of breast milk on day 5 was correlated with the total concentration of nitrite plus nitrate observed in breast milk on days 1 to 3. (5) The total concentration of nitrite plus nitrate in the breast milk of the high secretors significantly exceeded that seen in the low secretors. Conclusions:We suggest that nitric oxide is synthesized in the breast and may trigger lactation in humans. (J Pediatr 1997;131:839-43)

Application of the ILISPOT-IDIP system for the enumeration of different sizes of IgA spot forming cells in the murine small and large intestine

The immunofluorescence-linked immunospot (ILISPOT) assay associated with the immunofluorescence digital image processing (IDIP) system was originally developed in our laboratory to allow enumeration of immunoglobulin (Ig) producing, spot forming cells (SFC) in a more objective and quantitative manner. In this study, the ILISPOT-IDIP system was further advanced in order to analyze different sizes of SFC (e.g., IgA producing cells) including large (L), medium (M), and small (S) cells which correspond to high (> 2.4 pg), medium (1.2–2.4 pg) and low (< 1.2 pg) IgA secreting cells by the adaptation of real time image processor and intensified video camera system. When the ILISPOT-IDIP system was used to characterize the frequency of IgA secreting cells among mononuclear cells isolated from different parts of the murine gastrointestinal (GI) tract including the small (upper, middle and lower sections) and large (colon and rectum) intestine, the small intestine contained higher numbers of IgA SFC (∼ 8.4 × 10 5 SFC/10 6 cells) when compared with large intestine (∼ 1.3 × 10 5 SFC/10 6 cells). Among the 3 areas of small intestine, the upper (∼ 3.7 × 10 5 SFC/10 6 cells) and middle (∼ 2.4 × 10 5 SFC/10 6 cells) parts had higher numbers of IgA SFC when compared to the lower small (∼ 2.3 × 10 5 SFC/10 6 cells) intestine. When these IgA producing cells in different parts of the intestine were classified into three groups according to the size of individual spots, the upper and middle intestine contained higher frequencies of large (∼ 20%) and medium (∼ 20%) SFC which corresponded to high and medium IgA secretors in comparison to the lower small (∼ 9%) and large (∼ 6%) intestine. In contrast, the lower small and large intestine were dominated by small SFC since ∼ 85% of IgA producing cells were categorized as low secretors. Using the advanced ILISPOT-IDIP system, a unique distribution of different sizes (or secretion rates) of IgA producing SFC was elucidated in the different regions of the mouse small and large intestine.

Antimetastatic vaccination of tumor-bearing mice with two types of IFN-gamma gene-inserted tumor cells.

IFN-gamma genes were introduced through retroviral vectors into the high metastatic, low H-2Kb class I expressor, and poorly immunogenic 3LL-D122 clone. Two types of IFN-gamma infectants were isolated: IFN-gamma high secretors (128 to 256 IU/ml) and IFN-gamma non- or very low secretors (< 2 IU/ml). Both manifested high expression of MHC class I Ag. IFN-gamma secretors showed significant decrease in tumorigenicity and metastatic growth, whereas IFN-gamma nonsecretors retain tumorigenicity and were more metastatic than parental D122 cells. However, both groups, when inoculated in an irradiated form, induced similar high levels of CTL and protected mice to the same degree against a subsequent graft of parental cells. This indicates that enhanced expression of MHC class I and related genes caused by IFN-gamma is the major participant in CTL induction. Immunization of mice carrying an established tumor of parental D122 cells with IFN-gamma infectants is capable of almost completely preventing lung metastasis. Immunotherapy of tumor-bearing hosts is more effective when IFN-gamma secreting cells are used as immunogens, indicating that mechanisms, in addition to CTL, are stimulated by secreted IFN-gamma. Moreover, immunization with IFN-gamma high secretors of postoperative mice, carrying established micrometastases, almost completely cured these mice. Support for the participation of non-T cell effectors in the response to IFN-gamma secretors derives from the reduced tumorigenicity of these cells in CD1 nude mice. These data provide a rationale for the use of IFN-gamma gene-transferred tumor cells as a modality for cancer therapy.

Anti‐metastatic vaccination of tumor‐bearing mice with il‐2‐gene‐inserted tumor cells

IL-2 gene was introduced through retroviral vectors into the highly malignant and poorly immunogenic 3LL-D122 clone. Both high and low D122-IL-2 secretors showed elimination of tumorigenicity in syngeneic immune-competent mice; however, in nude mice only the high IL-2 secretor showed reduced tumorigenicity as compared with parental D122 cells. Also, following intravenous inoculation, only the high IL-2 secretor showed reduced generation of metastases, whereas the low IL-2 secretors were as highly metastatic as parental cells. These results seem to indicate that low levels of IL-2 secreted by tumor cells are sufficient to activate T cells, while higher levels are needed in order to activate non-T-cell effectors. D122-IL-2 secretors induced high levels of anti-tumor CTL, while their sensitivity to the lytic activity of these CTL was similar to the sensitivity of D122 cells, thus indicating that the elevation of immunogenicity of IL-2 secretors was essentially due to the secreted IL-2. In accordance with CTL induction, pre-immunization with IL-2 secretors protected mice against subsequent challenge of parental cells. Moreover, immunization in an "immunotherapy protocol" i.e., vaccination of mice carrying an established tumor of parental D122 cells with inactivated D122-IL2 infectants, almost completely eliminated the generation of lung metastases by D122 cells, thus providing a rationale for the use of IL-2 gene transferred tumor cells as a modality for treatment of metastasis.

HLA IN HUMAN SERUM—QUANTITATION OF CLASS I BY ENZYME IMMUNOASSAY 1

A solid-phase, enzyme-linked immunoassay was used to quantitate the soluble fraction of HLA-class I. The sera of 318 individuals were studied, as well as the urine of six individuals with normal renal function. The stability of blood concentrations of the soluble HLA was also evaluated. The data justify the following six conclusions. (1) All normal people have circulating HLA (mean = 357 ng/ml). (2) The population can be divided into one group of low secretors (mean = 162.4 +/- 65.2 ng/ml) and another group of high secretors (mean = 540.7 +/- 185.9 ng/ml) (P less than 0.01). (3) Blood levels in each individual are reasonably consistent over short (days) and long (years) periods of time. (4) The mean concentration of soluble HLA-class I in all renal failure patients was 590 ng/ml, significantly higher than normal (P = less than 0.05); it was highest in patients on peritoneal dialysis (mean = 683 ng/ml) in spite of substantial chronic loss in peritoneal dialysate. (5) Renal allograft recipients with stable allograft function also had mean values greater than normal at 554 ng/ml (P less than 0.05). (6) Soluble HLA-class I was not detected in the urine of individuals with normal renal function.

The Effect of HLA and Insulin‐Dependent Diabetes Mellitus on the Secretion Levels of Tumour Necrosis Factors Alpha and Beta and Gamma Interferon

Tumour necrosis factors alpha and beta (TNF-alpha and TNF-beta) and gamma interferon (IFN-gamma) were measured by ELISA in the supernatants of phytohaemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMNC) from 98 individuals (60 controls and 38 patients with insulin-dependent diabetes mellitus [IDDM]). The PBMNC were incubated with varying concentrations of PHA (0, 1, 5, and 10 micrograms/ml) for 72 h. In our population study we observed a correlation between the levels of secretion of TNF-alpha and IFN-gamma but not TNF-beta. The complete data set was analysed by non-parametric tests, and no associations with HLA phenotypes existed. Reduced levels of TNF-beta, but not TNF-alpha or IFN-gamma, secretion were found in IDDM patients stimulated with 1 and 5 micrograms/ml of PHA (P = 0.001 and 0.02 respectively). None of the lymphokine secretion levels at any PHA concentration correlated with particular HLA phenotypes. Analysis of the natural log-transformed data indicated that only for the TNF-beta levels (at 5 micrograms/ml PHA) could subjects be divided into high and low secretors, which also did not correlate with a particular HLA-B or -DR antigen.

Effect of bismuth subcitrate and sucralfate on rat duodenal and human gastric bicarbonate secretion in vivo.

Acid and alkali secretion have been examined together with prostaglandin E2 production in response to two mucosal protective drugs, colloidal bismuth subcitrate and sucralfate. Doses of colloidal bismuth subcitrate in the therapeutic range (120 and 1200 mg) had no effect on alkali secretion or luminal PGE2 output when perfused into the stomach of human volunteers. Similarly, in the anaesthetised rat, neither gastric acid nor duodenal alkali secretions were influenced by iv (12 mg/kg) or topical (120 mg/ml) administration of colloidal bismuth subcitrate. In contrast, perfusion of the human stomach with 1 g sucralfate stimulated bicarbonate output by 50%, a response which was unaffected by indomethacin (25 mg/h). A rise of 64% in gastric PGE2 output after sucralfate was, however, prevented by indomethacin pretreatment. Alkali secretion by rat duodenum was also increased by sucralfate but the response depended on the basal secretory rate. Low basal secretors (less than 3 mumol) showed a 75% stimulation whereas rats with high basal secretory rates (greater than 3 mumol) showed no significant response. All duodenal preparations regardless of basal secretory rate showed a stimulation of bicarbonate output with topical PGE2. The results suggest that enhancement of gastroduodenal bicarbonate secretion may play a role in the protective action of sucralfate but is unlikely to explain mucosal protection by colloidal bismuth subcitrate.

Factors affecting and patterns of residual insulin secretion during the first year of type 1 (insulin-dependent) diabetes mellitus in children.

We measured serum C-peptide, glucose, pH, islet antibodies and insulin antibody binding at diagnosis in 84 children with Type 1 (insulin-dependent) diabetes. In a subgroup of 33 children, residual insulin secretion (basal and peak C-peptide response to Sustacal), insulin antibody binding and HbA1c were measured at 10 days, 1, 3, 6 and 12 months. At presentation C-peptide correlated positively with age at onset and negatively with the blood glucose concentration. Median C-peptide concentration at diagnosis was low, rose significantly (p less than 0.05) at 10 days, reached a maximum at 1-3 months and declined gradually to 1 year. C-peptide concentration both at diagnosis and at 10 days correlated with that at 3 and 6 months. Of the factors investigated, only age (p less than 0.005) and sex (higher in females, p less than 0.01) were found to have a significant influence on basal/peak C-peptide levels throughout the first year. In particular there was no relationship between C-peptide, HbA1c and insulin dose during this period. A peak C-peptide response at 3-6 months greater than/less than 0.32 nmol/l was used to divide the group into two: 16 had a peak response less than 0.32 nmol/l (low secretors) while in 17, the peak C-peptide was greater than 0.32 nmol/l (high secretors). While the low secretors had significantly (p less than 0.05) lower C-peptide levels during the first year, there were no differences between low and high secretors in HbA1c or insulin dose.(ABSTRACT TRUNCATED AT 250 WORDS)

T cells with Fc receptors for IgA: induction of T alpha cells in vivo and in vitro by purified IgA.

Previous studies demonstrated that BALB/c mice with the IgA-secreting plasmacytomas MOPC-315 (alpha lambda 2), MOPC-167 (alpha kappa), McPC-603 (alpha kappa) and TEPC-15 (alpha kappa) developed large numbers of T cells with surface membrane receptors for IgA (T alpha cells). The lack of T alpha cell expansion in mice with variant plasmacytomas that were nonsecretors or low secretors of IgA implied that elevated serum IgA contributed to the increase in T alpha cells. The present studies show that normal BALB/c mice that were given daily injections of 30 mg of IgA (M315 protein) develop a marked increase in the number of T alpha cells. These studies also show that the T alpha cells induced by injection of IgA are Lyt1-2+ T cells. In addition, the data presented demonstrate that nylon wool nonadherent T cells, treated with purified polymeric IgA (M315 protein) in vitro, develop a marked increase in the number of T alpha cells. The in vitro induction of T alpha cells by IgA requires DNA and protein synthesis. These findings indicate that the T alpha cell expansion observed in mice with IgA myeloma is related to the high serum level of IgA and not to the myeloma tumor per se. In addition, these observations have a more general relevance to the issue of B cell regulation because they demonstrate that secreted immunoglobulin can directly induce expansion of immunoregulatory T cells.

Fibrinolytic activity in a human fibrosarcoma cell line and evidence for the induction of plasminogen activator secretion during tumor formation

Seven clones were isolated from the HT1080 human fibrosarcoma cell line using a fibrinagarose overlay technique. Three of these clones induced lysis of the fibrin overlay, whereas four did not. The extracellular and intracellular levels of protease were then measured using 125I-fibrin plates incubated with acid-treated human serum. The extracellular protease can be directly assayed in the medium from cells incubated with 10% fetal calf serum. Although there were large differences in the amounts of protease secreted by these two sets of clones, the intracellular levels of protease were similar. No significant differences were found between the abilities of the cells to grow in soft agar or as tumors in immunosuppressed hamsters. However, cells grown from tumors derived from all the low secretors of protease showed an increase in the amount of protease secreted. It appeared, therefore, that the secretion of protease might be selected for or induced during tumor growth. Further detailed studies with one of the low secreting clones (clone E) suggested an inductive rather than a selective mechanism for this increase in extracellular plasminogen activator.