Bestatin Treatment Research Articles

CD13 expression affects glioma patient survival and influences key functions of human glioblastoma cell lines in vitro

CD13 (APN) is an Alanyl-Aminopeptidase with diverse functions. The role of CD13 for gliomas is still unknown. In this study, data of glioma patients obtained by TCGA and CGGA databases were used to evaluate the survival rate and prognostic value of CD13 expression level. Protein expression of CD13 was confirmed by immunofluorescence staining of fresh patient tissues. Eight human glioblastoma cell lines were studied by RT-PCR, Western Blot, immunofluorescence staining and flow cytometry to define CD13 expression. Cell lines with different CD13 expression status were treated with a CD13 inhibitor, bestatin, and examined by MTT, scratch and colony formation assaysas well as by apoptosis assay and Western Blots. Bioinformatics analysis indicated that patients with high expression of CD13 had poor survival and prognosis. Additionally, CD13 protein expression was positively associated with clinical malignant characteristics. Investigated glioblastoma cell lines showed distinct expression levels and subcellular localization of CD13 with intracellular enrichment. Bestatin treatment reduced proliferation, migration and colony formation of glioma cells in a CD13-dependent manner while apoptosis was increased. In summary, CD13 has an impact on glioma patient survival and is important for the main function of specific glioma cells.

In Vivo Molecular Imaging of the Efficacy of Aminopeptidase N (APN/CD13) Receptor Inhibitor Treatment on Experimental Tumors Using 68Ga-NODAGA-c(NGR) Peptide.

Introduction The aminopeptidase N (APN/CD13) receptor plays an important role in the neoangiogenic process and metastatic tumor cell invasion. Clinical and preclinical studies reported that bestatin and actinonin are cytotoxic to APN/CD13-positive tumors and metastases due to their APN/CD13-specific inhibitor properties. Our previous studies have already shown that 68Ga-labeled NGR peptides bind specifically to APN/CD13 expressing tumor cells. The APN/CD13 specificity of 68Ga-NGR radiopharmaceuticals enables the following of the efficacy of antiangiogenic therapy with APN/CD13-specific inhibitors using positron emission tomography (PET). The aim of this in vivo study was to assess the antitumor effect of bestatin and actinonin treatment in subcutaneous transplanted HT1080 and B16-F10 tumor-bearing animal models using 68Ga-NODAGA-c(NGR). Materials and Methods Three days after the inoculation of HT1080 and B16-F10 cells, mice were treated with intraperitoneal injection of bestatin (15 mg/kg) or actinonin (5 mg/kg) for 7 days. On the 5th and 10th day, in vivo PET scans and ex vivo biodistribution studies were performed 90 min after intravenous injection of 5.5 ± 0.2 MBq68Ga-NODAGA-c(NGR). Results Control-untreated HT1080 and B16-F10 tumors were clearly visualized by the APN/CD13-specific 68Ga-NODAGA-c(NGR) radiopharmaceutical. The western blot analysis also confirmed the strong APN/CD13 positivity in the investigated tumors. We found significantly (p ≤ 0.05) lower radiopharmaceutical uptake after bestatin treatment and higher radiotracer accumulation in the actinonin-treated HT1080 tumors. In contrast, significantly lower (p ≤ 0.01) 68Ga-NODAGA-c(NGR) accumulation was observed in both bestatin- and actinonin-treated B16-F10 melanoma tumors compared to the untreated-control tumors. Bestatin inhibited tumor growth and 68Ga-NODAGA-c(NGR) uptake in both tumor models. Conclusion The bestatin treatment is suitable for suppressing the neoangiogenic process and APN/CD13 expression of experimental HT1080 and B16-F10 tumors; furthermore, 68Ga-NODAGA-c(NGR) is an applicable radiotracer for the in vivo monitoring of the efficacy of the APN/CD13 inhibition-based anticancer therapies.

Abstract 16: LTA4 hydrolase inhibitor bestatin prevents intestinal tumors in APC mutant rodent models of FAP

Abstract Colorectal cancer (CRC) is the third most common cancer in the United States with 145,600 new cases and 51,020 deaths estimated to occur in 2019. Leukotriene B4 (LTB4), the product of the enzyme leukotriene A4 hydrolase (LTA4H), reportedly plays a role in carcinogenesis. LTA4H over expression and increased LTB4 levels are found in CRC tissues and correlate with both disease stage and reduced survival. Thus, we used a preclinical rodent model to assess repurposing bestatin (Ubenimex), a potent orally-active reversible small molecule inhibitor of LTA4H, for prevention of CRC in high-risk FAP patients. Six week old Apcmin/+ male and female mice (n≥15) were assigned to control and treatment groups; groups received the same diet either with 1000 ppm bestatin (treatment) or no bestatin (control) for 14 weeks at which point mice were euthanized and intestines were evaluated for tumors. Control mice developed colonic tumors with multiplicity of 1.35±0.16 (Mean±SEM) in males and 0.86±0.13 in females. Colonic tumor incidence was 91% and 73% in the male and female mice respectively. Additionally, control mice developed 30.52±1.66 (male) and 29.57±2.74 (female) small intestinal polyps (SIP). Dietary bestatin significantly reduced colon tumor multiplicity. Bestatin treated male mice had 55% fewer colonic tumors (0.60±0.21; p<0.01) and female mice 59% fewer (0.35±0.12; p<0.008) compared to their respective controls. Importantly colonic tumor incidence was inhibited by >50% in both genders. Bestatin administration was reduced colonic tumor incidence by 56% (p<0.001) in male and 52% (p<0.026) in female mice. Small intestinal polyps were also significantly reduced in treated mice compared to controls. Male mice developed 29% less SIP (21.67±2.32; p<0.006) while female had 46% fewer SIP (16.00±1.98; p<0.0005) compared to controls. The polyposis in rat coli (PIRC) model replicates CRC in FAP patients. PIRC rats (n=6) at 8 weeks of age were assessed for polyp number by colonoscopy, and assigned to control and bestatin (1000 ppm) groups for 8 weeks. Colonic polyp inhibition in each rat was evaluated by colonoscopy after treatment. Bestatin treatment led to strong suppression of colonic polyps; significantly fewer (~72% less; p<0.005) colonic polyps were seen in bestatin-treated PIRC rats. Pharmacokinetic analysis demonstrated that dietary bestatin resulted in significant bestatin levels in both serum (15μg/mL) and colonic tissue (up to 250 μg/g). LTB4 levels in both plasma and colon tumors were also significantly lowered by ~70% (p<0.004) and ~26% (p<0.01) respectively. Colonic tumors from the treated animals also showed an increase in NK and NKT cells suggesting immune-modulatory effects of the agent. In summary, bestatin demonstrated excellent safety, pharmacokinetics, and chemopreventive effects against CRC in preclinical models and warrants further validation clinically. Citation Format: Venkateshwar Madka, Gopal Pathuri, Hariprasad Gali, Nandini Kumar, Nagendra Sastri Yarla, Anh Bao, Adam S. Asch, Chinthalapally V. Rao. LTA4 hydrolase inhibitor bestatin prevents intestinal tumors in APC mutant rodent models of FAP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 16.

The Activity of a Hexameric M17 Metallo-Aminopeptidase Is Associated With Survival of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is one of the most prevalent human pathogens causing millions of deaths in the last years. Moreover, tuberculosis (TB) treatment has become increasingly challenging owing to the emergence of multidrug resistant M. tuberculosis strains. Thus, there is an immediate need for the development of new anti-TB drugs. Proteases appear to be a promising approach and may lead to shortened and effective treatments for drug-resistant TB. Although the M. tuberculosis genome predicts more than 100 genes encoding proteases, only a few of them have been studied. Aminopeptidases constitute a set of proteases that selectively remove amino acids from the N-terminus of proteins and peptides and may act as virulence factors, essential for survival and maintenance of many microbial pathogens. Here, we characterized a leucine aminopeptidase of M. tuberculosis (MtLAP) as a cytosolic oligomeric metallo-aminopeptidase. Molecular and enzymatic properties lead us to classify MtLAP as a typical member of the peptidase family M17. Furthermore, the aminopeptidase inhibitor bestatin strongly inhibited MtLAP activity, in vitro M. tuberculosis growth and macrophage infection. In murine model of TB, bestatin treatment reduced bacterial burden and lesion in the lungs of infected mice. Thus, our data suggest that MtLAP participates in important metabolic pathways of M. tuberculosis necessary for its survival and virulence and consequently may be a promising target for new anti-TB drugs.

Protective effects of bestatin in the retina of streptozotocin-induced diabetic mice

CD13/APN (aminopeptidase N) was first identified as a selective angiogenic marker expressed in tumor vasculature and is considered a target for anti-cancer therapy. CD13 was also reported to express in non-diabetic, hypoxia-induced retinal neovascularization. Whether diabetes induces upregulation of CD13 expression in the retina is unknown. We hypothesize that at an early stage of non-proliferative diabetic retinopathy (NPDR) characterized by disruption of blood-retinal barrier (BRB) permeability is related to upregulated expression of CD13 because of its known role in extracellular matrix (ECM) degradation. The purpose of this study is to evaluate the role of CD13/APN and the therapeutic efficacy of a CD13/APN inhibitor in a mouse model of streptozotocin-induced NPDR. Hyperglycemic C57Bl/6 mice 26 weeks after streptozotocin (STZ) injection were intravitreally injected with a sustained release formulation of CD13/APN inhibitor bestatin. At 15th day of post-bestatin treatment, mouse retinas were evaluated for vascular permeability by Evans blue dye extravasation assay, fluorescent angiography of retinal vascular permeability and leukostasis. Retinal protein extracts were analyzed by Western blot to determine the effects of bestatin treatment on the expression of CD13/APN related inflammatory mediators of ECM degradation and angiogenesis. Intravitreal bestatin treatment significantly inhibited retinal vascular permeability and leukostasis. This treatment also significantly inhibited retinal expression of CD13, ECM degrading proteases (heparanase and MMP9 and angiogenic molecules (HIF-1α and VEGF). Intravitreal CD13 inhibition may relate to furthering our knowledge on the protective effect of bestatin against diabetic retinal vasculature abnormalities through inhibition of retinal permeability, leukostasis, inflammatory molecules of ECM degradation and angiogenesis.

Possible contribution of aminopeptidase N(APN/CD13) to migration and invasion of human osteosarcoma cell lines.

Osteosarcoma is the most common primary malignancy of the bone. Aminopeptidase N (APN/CD13), a Zn+2-dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion mechanism. This study explores the potential involvement of APN in migration and invasion of human osteosarcoma cells in vitro using inhi-bitors and activators of APN. Cells treated with APN inhibitor bestatin displayed decreased migration and invasion in a Boyden chamber Transwell assay. Western blotting revealed reduced levels of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathway proteins, reduced phosphorylation of p38, ERK1/2 and JNK and decreased levels of NF-κB. Bestatin treatment also lowered APN, matrix metalloproteinase (MMP)-2 and -9 enzymatic activity and their mRNA expression. Reduced MMP-2 and -9 protein levels were also observed. By comparison, cells treated with cytokine interleukin-6 (IL-6), a stimulator of APN, displayed increased migration and invasion. Western blotting revealed increased levels of MAPK and PI3K pathway proteins, phosphorylated p38, ERK1/2 and JNK, and NF-κB. IL-6 treatment also increased APN and MMP-2 and -9 enzymatic activity. An increase of APN, MMP-2 and -9 mRNA levels, and MMP-2 and -9 protein levels was also observed. Together these experiments reveal potential enzymatic and signalling roles for APN in osteosarcoma and establish a starting point for an in-depth analysis of the role of APN in regulating invasiveness. A deeper knowledge about the regulatory mechanisms of APN may contribute to the development of anti-metastatic therapies.

Leukemic cell-surface CD13/aminopeptidase N and resistance to apoptosis mediated by endothelial cells.

Attachment of leukemic cells to vascular endothelial cells induces the vascular endothelial cells to release endothelial cell-derived interleukin 8 (endothelial IL-8), which then induces leukemic cells to undergo apoptosis. NB4, a human promyelocytic leukemic cell line that expresses high levels of cell-surface CD13/aminopeptidase N, does not undergo endothelial IL-8-induced apoptosis. Consequently, we investigated the relationship between cell-surface aminopeptidase activity and endothelial IL-8 induction of apoptosis in various leukemic cell lines. CD13/aminopeptidase N activity and IL-8-induced apoptosis were examined in leukemic cell lines. Endothelial IL-8-induced apoptosis was examined further in NB4 cells, K562 cells (human chronic myelogenous leukemic cells expressing low levels of CD13/aminopeptidase N), CD13/aminopeptidase N-transfected K562 (K562/CD13) cells that overexpress aminopeptidase, and mock-transfected K562 cells (vector only). These cells were also cocultured with a vascular endothelial cell layer to investigate the association between aminopeptidase activity and apoptosis in this system. All statistical tests were two-sided. Endothelial IL-8 induced apoptosis in K562 cells but not in K562/CD13 cells. A combination of an aminopeptidase inhibitor (such as bestatin) and endothelial IL-8 induced apoptosis in NB4 cells and K562/CD13 cells (2.88-fold difference [95% confidence interval [CI] = 1.82-fold to 3.94-fold], P =.004 for bestatin-treated NB4 cells and 4.31-fold difference [95% CI = 3.52-fold to 5.10-fold], P<.001 for bestatin-treated K562/CD13 cells). When aminopeptidase activity in NB4 cells was modulated by aminopeptidase inhibitors, a statistically significant correlation was found between aminopeptidase activity and the proportion of apoptotic cells induced by endothelial IL-8 (r = -.837, P<.001 by Pearson's correlation coefficient; r = -.697, P =.013 by Spearman's correlation analysis by ranks). K562/CD13 cells cocultured with vascular endothelial cells did not undergo apoptosis, but the addition of bestatin resulted in the induction of apoptosis in K562/CD13 cells (2.70-fold difference [95% CI = 1.77-fold to 3.63-fold], P<.001). Bestatin treatment increased the level of IL-8 mRNA in and the amount of IL-8 secreted by vascular endothelial cells. High levels of cell-surface CD13/aminopeptidase N appear to allow leukemic cells to resist endothelial IL-8-induced apoptosis. The combination of endothelial IL-8 and bestatin induce leukemic cells expressing high levels of CD13/aminopeptidase N to undergo apoptosis. Bestatin may be useful for treating patients with leukemia.

Effect of Ubenimex (Bestatin) on The Cell Growth and Phenotype of HL-60 and HL-60R Cell Lines: Up-and Down-Regulation of CD13/Aminopeptidase N

Ubenimex (Bestatin), a low-molecular-mass dipeptide, has been demonstrated to have anti-tumor activities and immunomodulating activities. We here report cell growth inhibition and phenotypic changes of HL-60 and HL-60R cell lines induced by Bestatin treatment. Bestatin (0.1 m¨g/ml) showed remarkable cell growth inhibition against HL-60 cells, whereas it was ineffective for HL 60R cells. Bestatin also showed growth inhibition in the concentration of 1 m¨ g/ml against HL-60R cells which are resistant to differentiation induction by DMSO and TPA. In both cell types, the effect of growth inhibition by Bestatin treatment was dose dependent.We found a low level expression of CD13 and a low number of CD13 positive cells in HL-60R cells compared with that of HL-60. We also observed phenotypic changes of HL-60 and HL-60R cells following incubation with Bestatin (10 m¨g/ml) for 1 and 3 hrs, respectively. With HL-60 cells, the upregulation of CD13/aminopeptidase N was found after 1 hr, however, the downregulation was observed after 3 hrs incubation with Bestatin. On the other hand, the downregulation of CD 15 and CD33 was observed after both one and 3 hrs incubation. Similarly, in HL-60R cells, the upregulation of CD13/aminopeptidase N was found temporarily (1hr), and then CD13 downregulation was observed after 3 hrs incubation with Bestatin. No notable change was observed for expression of other myeloid-related antigens, e.g. CD14 (My4, LeuM3), CDllb (OKM1), and CD34 (My10). On the basis of these observations of in vitro activity, we suggest that Bestatin may also be an effective anti-leukemic agent in vivo.

Growth-stimulating phase of macrophage response to activation: the phenomenon and its implications for tumour growth and immunotherapy.

The dynamics of growth-stimulating and cytotoxic activity of mouse peritoneal macrophages (PMo) in response to in vivo and in vitro bacillus Calmette-Guérin (BCG) or bestatin treatment was studied. It was shown that BCG and bestatin induce cytotoxicity in PMo, and that after the cytotoxic response strong growth-stimulating activity develops. PMo, rendered cytotoxic in vivo and afterwards cultivated in vitro, displayed the same switch from a cytotoxic to a growth-stimulating phase. These results suggest that the growth-stimulating phase is the obligatory PMo response to biological response modifiers (BRM) at least to BCG and bestatin. The growth rate of tumours, transplanted into mice during the cytotoxic phase of the response to BCG, was suppressed, whereas tumours transplanted during the growth-stimulating phase were stimulated. It appears that the development of a growth-stimulating phase after the cytotoxic phase of response to activation by BRM could be one of the reasons for the limited effectiveness of immunotherapy based on the application of macrophage activators.

Bestatin treatment of myelodysplastic syndromes and chronic myelogenous leukemia

A high remission rate (56%) was achieved in a preliminary study using Bestatin in patients with myelodysplastic syndromes. In particular, 9 out of 13 patients (69%) in the high blast group achieved hematologic remission. After Bestatin treatment, intrinsic hematopoietic stem cell abnormalities as well as hematologic findings were markedly improved. The success of Bestatin therapy in MDS led us to investigate the clinical activity of Bestatin in CML. In the current study the busulfan and Bestatin combination therapy resulted in complete hematologic remission in all of the patients. The most exciting result was the suppression of the Philadelphia chromosomes among the responding patients. Complete cytogenetic response was obtained in 3 patients (21%), partial cytogenetic response in 1 (7%), and minor cytogenetic response in 5 (36%). In particular, the majority of early chronic phase CML patients achieved significant cytogenetic response with sustained Ph 1 negativity. The results are very encouraging and warrant further studies. myelodysplastic syndromes/chronic myelogenous leukemia/Bestatin biotherapy

Bestatin administration and the change in cellular immunity

The change in cellular immunity by Bestatin (ubenimex) treatment — 30 mg/day orally — was investigated in 23 gastrointestinal cancer patients for: 1), functional T cell subsets; 2), IL-2 receptor; 3), PHA-induced blastogenesis; and 4), PPD skin reaction. The absolute number of helper T cells (T h) and cytotoxic T cells (T c) increased in 74 and 79% of cases, respectively, compared with pretreatment values. On the other hand, the absolute number of suppressor T cells (T s) decreased in 79% of cases. IL-2 receptor increased in 56% of patients, PHA blastogenesis increased in 67% of patients and PPD skin reaction was elevated in 75% of cases comparing to pretreatment values. These results suggest that Bestatin could increase cellular immunity in cancer patients. Bestatin (ubenimex)/clinical use/mechanism

Bestatin Treatment of Myelodysplastic Syndromes (MDS) and the Effects of Bestatin on Hematopoiesis in MDS

A high remission rate (75%) was achieved in a preliminary study using bestatin in patients with refractory anemia with excess blasts (RAEB) and RAEB in transformation (RAEB-t). One of 2 patients with RAEB-t and 3 of 6 patients with RAEB obtained complete response. Two patients with RAEB achieved good response, but one with refractory anemia failed. Clonogenic marrow cell culture studies in patients with myelodysplastic syndromes have demonstrated intrinsic hematopoietic stem cell abnormalities, in particular defective erythroid colony formation. After bestatin treatment, these abnormalities as well as hematologic findings were markedly improved. The results suggest that bestatin has an enhancing effect on burst promoting activity production of helper (CD4 positive) T lymphocytes and the effect on hematopoiesis of bestatin may be mediated by T lymphocytes.

Adjuvant Bestatin (Ubenimex) Treatment Following Full-Dose Local Irradiation for Bladder Carcinoma

The clinical value of adjuvant bestatin (Ubenimex) immunotherapy has been examined in a group of patients with urinary bladder cancer. Patients with non-metastatic transitional cell carcinoma of the bladder, scheduled for full-dose local irradiation therapy (64 Gy), were randomly allocated to adjuvant oral bestatin treatment (30 mg daily for at least 1 year), starting at completion of irradiation, or no bestatin. The trial included 194 evaluable patients with a follow-up period of 1.5-9.5 years. The overall survival of the two groups of patients did not differ statistically significantly (97 patients in each). Subgrouping of the patient material gave no evidence that the clinical efficacy of bestatin is related to sex, tumor category or malignancy grade.

From experimental to clinical attempts in immunorestoration with bestatin and zinc

Severe impairment of the lymphopoietic cell renewal system is an important etiological factor of cancer development and it may be the consequence of massive radio and/or chemotherapeutic regimens. In a comparative study, we analysed the potential, systemic immunorestoratory capacity of bestatin, a microbial leucil-aminopeptidase inhibitor and of the ubiquitous trace element zinc. In vivo administration of bestatin in mice stimulated both Interleukin 1 and Interleukin 2 production, and enhanced T cell, B cell as well as macrophage mediated immuno-reactions. In a phase II clinical trial on 41 patients with non-Hodgkin lymphoma, Hodgkin disease and solid tumors, bestatin treatment corrected the pathological frequency of both OKT4 and OKT8 lymphocyte subpopulations. Zinc-saturated transferrin had a significative stimulatory effect on the ongoing DNA synthesis of antigen activated human lymphocytes in culture. Oral administration of zinc-gluconate to patients who manifested a severe T cell subpopulation defect corrected preferentially the OKT8 suppressor/cytotoxic T cell unbalances. The clinical results obtained by both bestatin and zinc were observed only on a short-term, so further studies are needed to elaborate long lasting regiments and to establish whether these treatments have determinant influence on the underlying disease.

Cells responsible for tumor surveillance in man: effects of radiotherapy, chemotherapy, and biologic response modifiers.

Currently, the most probable theory of tumor surveillance is neither the existence of any tumor-specific, antigen-dependent, T-cell-mediated cytotoxic effect that could eliminate spontaneous tumors in man and that could be used for some kind of vaccination against tumors, nor the complete absence of any surveillance or defense systems against tumors. What is probable is the cooperation of a number of antigen-independent, relatively weakly cytotoxic or possibly only cytostatic humoral and cellular effects, including nutritional immunity, tumor necrosis factor, certain cytokines, and the cytotoxic effects mediated by macrophages, NK cells, NK-like cells, and certain stimulated T-cells. One question remaining to be solved is why these antigen-independent effects do not attack normal cells. A number of plausible hypotheses are discussed. The hypothetical surveillance system is modulated both by traditional cancer treatment and by attempts at immunomodulation. Radiotherapy reduced the T-helper cell function for almost a decade, but not those of macrophages or NK cells. T-cell changes have no prognostic implication, supporting, perhaps, the suggestion of a major role for macrophages and NK cells. Cyclic adjuvant chemotherapy reduces the peripheral lymphocyte population and several lymphocyte functions but not NK activity. Most of the parameters were normalized some years following treatment, but NK activity remained elevated and Th/Ts cell ratio was still decreased. This might possibly be taken to support the surveillance role of NK cells. Bestatin increases the frequency of lymphocytes forming rosettes with sheep red blood cells (but not their mitogenic responses), enhances NK activity, and augments the phagocytic capacity of granulocytes and monocytes (but not their cytotoxic activity). Improved survival with Bestatin treatment following chemotherapy has been observed in patients with melanoma Stages 1b and II and in patients with acute nonlymphatic leukemia, where BCG also seems active, although possibly only in patient groups with less than 49% complete remissions.

Bestatin treatment for the correction of granulocyte dysfunction in patients with recurrent furunculosis.

Bestatin, a new immunomodulator which is chemically well-defined, was examined for its capacity to enhance the phagocytic activity of neutrophilic granulocytes from patients with furunculosis. The ability of the granulocytes to ingest fluorescein-labelled yeast particles was significantly decreased in 19 patients with recurrent furunculosis (p less than 0.01). Oral administration of 40 mg bestatin to ten patients increased the phagocytic function of their granulocytes significantly (p less than 0.01).

Bestatin treatment of human lymphocytes increases the frequency of sheep red blood cell-binding cells

Treatment of cancer patients with Bestatin, a new immunomodulator increases the frequency of peripheral lymphocytes forming rosettes with sheep red blood cells (SRBC). This study has shown that treatment of lymphocytes from healthy donors with Bestatin in vitro increases the frequency of such cells and there is a slight decrease of C'3-receptor bearing cells. The proportions of T M and T G cells were not significantly changed. The results indicate that the increased frequency of SRBC-binding cells during Bestatin medication is not due to a mobilisation of T-cells but rather to an improved binding capacity of certain T-cells for SRBC.

Influence of Bestatin, a new immunomodulator, on Ig secretion by human lymphocytes in vitro and in vivo.

Bestatin, a chemically well-defined immunomodulator, was tested for its capacity to interfere with PWM-induced Ig synthesis of peripheral lymphocytes from healthy donors. It was observed that secretion of IgA, IgG and IgM was impaired when Bestatin was present in the cultures. Serum Ig concentrations and antibody activities of IgG class to cytomegalovirus, herpes simplex and morbilli virus were examined in cancer patients who were treated with 30 mg of Bestatin daily for 1-21 weeks. These values remained constant during Bestatin treatment indicating that antibody secretion in vivo was not affected.

Bestatin treatment and the peripheral lymphocyte population in cancer patients.

Bestatin, a substance produced by Streptomyces olivoreticuli, inhibits certain cell-membrane-associated enzymes and has been shown to augment immune responses in experimental animals. We have determined whether bestatin medication changed the peripheral lymphocyte population in 15 advanced cancer patients. After 2 weeks of daily, 30 mg oral bestatin medication, the lymphocyte counts remained essentially unchanged, but the frequency of E-rosette-forming lymphocytes increased. In vitro stimulation of the lymphocytes with PHA or PPD remained essentially unchanged while the natural-killer activity of the lymphocyte population increased in most patients. Bestatin treatment caused no detectable side effects.

Mitogenic effect of bestatin on lymphocytes.

The incorporation of 3H-thymidine into the acid insoluble fraction of spleen cells was increased intraperitoneal injection of 10 microgram bestatin/mouse. Bestatin (0.01, 0.1, 1.0 microgram/ml) which was added to mouse spleen cell culture increased 3H-thymidine incorporation into lymphocytes, but 10 microgram/ml did not. This mitogenic action of bestatin was not observed when adherent cells were removed or T cells were destroyed, suggesting that bestatin causes the proliferate T cells probably through the activation of macrophages. Bestatin given to mice did not modulate mitogenicity of lectins. Bestatin treatment of human peripheral buffy coat cells increased 3H-thymidine incorporation into lymphocytes. The addition of bestatin at a high concentration such as 100 microgram/ml to mouse spleen cell cultures exhibited a high mitogenic effect on B cells in preference to T cells but this effect was not seen with bestatin at 50 microgram/ml. In this case, the mitogenicity of Con A or LPS was expanded and antibody formation to SRBC in spleen cell cultures were also stimulated.