Effect of adjuvant trastuzumab treatment in conventional clinical setting: an observational retrospective multicenter Italian study.

Clinical trials have shown the efficacy of trastuzumab-based adjuvant therapy in HER2-positive breast cancers, but routine clinical use awaits evaluation of compliance, safety, and effectiveness. Adjuvant trastuzumab-based therapy in routine clinical use was evaluated in the retrospective study GHEA, recording 1,002 patients treated according to the HERA protocol between March 2005 and December 2009 in 42 Italian oncology departments; 874 (87.23 %) patients completed 1-year trastuzumab treatment. In 128 patients (12.77 %), trastuzumab was withdrawn due to cardiac or non-cardiac toxicity (28 and 29 patients, respectively), disease progression (5 patients) or the clinician's decision (66 patients). In addition, 156 patients experienced minor non-cardiac toxicities; 10 and 44 patients showed CHF and decreased LVEF, respectively, at the end of treatment. Compliance and safety of adjuvant trastuzumab-based therapy in Italian hospitals were high and close to those reported in the HERA trial. With a median follow-up of 32 months, 107 breast cancer relapses were recorded (overall frequency, 10.67 %), and lymph node involvement, estrogen receptor negativity, lymphoid infiltration, and vascular invasion were identified as independent prognostic factors for tumor recurrence, indicating that relapses were associated with advanced tumor stage. Analysis of site and frequency of distant metastases showed that bone metastases were significantly more frequent during or immediately after trastuzumab (<18 months from the start of treatment) compared to recurrences in bone after the end of treatment and wash-out of the drug (>18 months from the start of treatment) (35.89 vs. 14.28 %, p = 0.0240); no significant differences were observed in recurrences in the other recorded body sites, raising the possibility that the protection exerted by trastuzumab is lower in bone metastases.

Regulation of the macrophage content of neoplasms by chemoattractants.

Factor chemotactic for mononuclear phagocytes was found in supernatant fluids of cultured human and mouse tumor cells. In 11 mouse tumors there was a correlation observed between chemotactic activity and macrophage content of neoplastic tissues. Tumor-derived chemoattractants appear to participate in the regulation of tumor-associated macrophages.

Biology, prognosis and response to therapy of breast carcinomas according to HER2 score.

BACKGROUND: The standardization of the HER2 score and recent changes in therapeutic modalities points to the need for a reevaluation of the role of HER2 in recently diagnosed breast carcinoma. PATIENTS AND METHODS: A multicenter, retrospective study of 1794 primary breast carcinomas diagnosed in Italy in 2000/2001 and scored in HER2 four categories according to immunohistochemistry was conducted. RESULTS: Ductal histotype, vascular invasion, grade, MIB1 positivity, estrogen and progesterone receptor expression differed significantly in HER2 3+ tumors compared with the other categories. HER2 2+ tumors almost showed values intermediate between those of the negative and the 3+ subgroups. The characteristics of HER2 1+ tumors were found to be in between those of HER2 0 and 2+ tumors. With a median follow-up of 54 months, HER2 3+ status was associated with higher relapse rates in node-positive and node-negative subgroups, while HER2 2+ only in node positive. Analysis of relapses according to type of therapy provided evidence of responsiveness of HER2-positive tumors to chemotherapy, especially taxanes. CONCLUSIONS: The present prognostic significance of HER2 is correlated to receptor expression level and points to the need to consider HER2 2+ and HER2 3+ tumors as distinct diseases with different outcomes and specific features.

Contribution of CD4+, CD8+CD28+, and CD8+CD28- T cells to CD3+ lymphocyte homeostasis during the natural course of HIV-1 infection.

The relationship between the number of circulating CD4+ T cells and the presence of particular CD8+ T cell subsets was analyzed by flow cytometry on PBL from asymptomatic HIV-1-infected patients whose specimens were collected every 2 mo for a total period of 32 mo. Only slight variations were detected in the absolute number of lymphocytes and percentage of CD3+ lymphocytes, whereas both CD4+ and CD8+ T cell subsets showed wide intrapatient variation. Variations in the number of CD8+CD28+ cells paralleled those of the CD4+ T cell subset in each patient tested, while the presence of CD8+CD28- T cells correlated inversely with CD4+ and CD8+CD28+ T cells. These data show that changes in the number of circulating CD4+-and CD8+CD28+ T cells are strongly related to the presence of CD8+CD28- T cells in these patients. Insight into the significance of CD8+CD28- T cell expansion will allow us to understand the mechanisms and significance of the HIV-1- driven change in CD4+CD8+ T cell homeostasis and the basic immunopathology of HIV disease.

Role of exon-16-deleted HER2 in breast carcinomas.

A splice variant of the human gene HER2, lacking exon-16 (DeltaHER2) which encodes a small extracellular region, has been described. This altered receptor forms disulfide bond-stabilized homodimers. We report here that the DeltaHER2 splice variant represents about 9% of the HER2 mRNA obtained from most of the 46 breast carcinoma samples with HER2 expression levels ranging from 3+ to 0 by HercepTest. Analysis of human cells transfected with DeltaHER2 or wild-type (WT) cDNA revealed no growth of WT cells in nude mice, whereas clones expressing 10-fold less DeltaHER2 were tumorigenic. Unlike WT transfectants, DeltaHER2-expressing cells showed low sensitivity to two new therapeutic drugs targeting receptors of the HER family (ZD1839 and Trastuzumab), whereas an inhibitor of the HER2 tyrosine kinase domain (Emodin) blocked activation of both DeltaHER2 and WT transfectants. Taken together, our findings indicate that the DeltaHER2 transcript encodes the transforming form of the oncoprotein. It is plausible that malignant transformation arises when a critical threshold of DeltaHER2 is reached in HER2-overexpressing tumors. Specific inhibitors of HER2 catalytic activity represent a promising approach to therapy of HER2-overexpressing tumors.

Human renal antigen defined by a murine monoclonal antibody.

Fusion of spleen cells from a mouse immunized with a surgical specimen of a human renal carcinoma with murine P3 myeloma cells resulted in the establishment of a hybridoma cell line that secreted a monoclonal antibody (MKi-1), of IgG1 subclass, which preferentially reacted on kidney crude membrane (CM) preparations. This monoclonal antibody was tested by solid-phase radioimmunometric assay and immunofluorescence (IF) on a panel of tumor cell lines and on CM preparations and cell suspensions from surgical specimens of normal and neoplastic tissues. In addition, cryosections of normal and cancer tissues of various histologic types were tested by IF. The expression of the MKi-1 antigen was limited to normal kidney epithelium, renal cancers, some areas in the pancreas, the apical region of some breast ducts, and a proportion (5-50%) of activated lymphocytes. Electron microscopic study by the immunoperoxidase technique on fixed sections from normal kidney showed that MKi-1 stained the brush border of almost all proximal tubules. The molecule recognized by MKi-1 was a single polypeptide chain with a molecular weight of 140,000.

Oral administration of anti-doxorubicin monoclonal antibody prevents chemotherapy-induced gastrointestinal toxicity in mice.

Gastrointestinal mucositis is a common and painful condition that afflicts a proportion of cancer patients receiving chemotherapeutic drugs including anthracyclines, and it has become the dose-limiting toxicity for a number of chemotherapeutic regimens. The murine monoclonal antibody MAD11 recognizes the anthracycline doxorubicin, and systemic administration of this antibody in mice treated with doxorubicin was found previously to prevent the toxic effects of the drug. The purpose of this study was to determine whether gastrointestinal toxicity associated with doxorubicin can be reduced by oral administration of anti-doxorubicin MAD11 in mice. Our experiments show that orally administered MAD11 antibodies: (a) are essentially not absorbed in the blood circulation since less than 0.5% of protein-associated radioactivity was recovered from blood samples; (b) reduce the extent of doxorubicin-induced apoptosis in murine intestinal crypts, as determined by labeling strand breaks with modified nucleotides in an enzymatic reaction; and (c) reduce the body weight loss in mice treated with 12 mg/kg body weight of doxorubicin and decrease the early mortality in mice treated with 16 mg/kg body weight. This type of treatment may be useful in preventing anthracycline-induced gastrointestinal mucositis in cancer patients.

Immunochemical analysis of the determinant recognized by a monoclonal antibody (MBr1) which specifically binds to human mammary epithelial cells.

A monoclonal antibody (MBr1) raised against a membrane preparation (CM) of a human breast cancer line (MCF-7) and characterized as mammary gland epithelium associated (S. Mènard, E. Tagliabue, S. Canevari, G. Fossati, and M. I. Colnaghi. Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res., 43:1295-1300, 1983), was used to biochemically define and partially purify its target antigen. The antigenic activity recognized by MBr1 was unaffected by treatment of MCF-7 cells with trypsin, protease K, or Vibrio cholerae neuraminidase and by heating at 100 degrees but was abolished by treatment with methanol. Since this behavior suggested a glycolipid nature of the MBr1-defined antigen, total lipids were obtained by chloroform:methanol or tetrahydrofuran:phosphate buffer extractions from crude membrane preparations of MCF-7 cells and of breast cancer surgical specimens. Total absorption of MBr1 activity was found by breast cancer lipid extracts, whereas no absorbing capability was detected with a series of highly purified acid and neutral glycolipids or with normal and neuraminidase-treated red blood cells of human, ox, and sheep species. The same pattern of inhibition of MBr1-binding activity was obtained with total lipid extract and both phases after diethyl ether partition. However, when the three extracts were chromatographed on diethylaminoethyl-Sepharose, the antigenic activity was recovered only in the neutral glycolipid fractions. Periodate oxidation of MCF-7 crude membrane preparation abolished MBr1-binding activity, suggesting that the carbohydrate portion of the molecule may constitute the antigenic determinant.

Cooperative effects of Mycobacterium tuberculosis Ag38 gene transduction and interleukin 12 in vaccination against spontaneous tumor development in proto-neu transgenic mice.

An approach to stimulating an immune response against tumors is to transduce tumor cells with bacterial genes, which represent a "danger signal" and can induce a wide immune response. Mycobacterium tuberculosis genes and their encoded proteins play a pivotal role in linking innate and cell-mediated adaptive immunity and represent ideal candidates as immune adjuvants for tumor vaccines. The efficacy of a cancer vaccine, obtained by transduction of a mammary tumor cell line with the M. tuberculosis Ag38 gene, was investigated in female mice transgenically expressing the rat HER-2/neu proto-oncogene. These mice spontaneously develop stochastic mammary tumors after a long latency period. The onset of spontaneous mammary tumors was significantly delayed in mice vaccinated with Ag38-transduced cells but not in mice vaccinated with nontransduced cells as compared with untreated mice. Protection from spontaneous tumor development was increased when mice were vaccinated with the mycobacterium gene-transduced vaccine plus a systemic administration of interleukin 12 (IL-12) at a low dose. Mice vaccinated with nontransduced cells plus IL-12 developed tumors, with only a slight delay in tumor appearance as compared with the control group. Lymphocytes obtained from lymph nodes of mice vaccinated with transduced cells secreted high levels of IFN-gamma. CD3+CD8+ spleen cells derived from these mice responded to the tumor with IFN-gamma production. These data indicate the efficacy of a short-term protocol of vaccinations exploiting the adjuvant potency of a M. tuberculosis gene and low doses of IL-12 in a model of stochastic development of mammary tumors. This adjuvant approach may represent a promising immunotherapeutic strategy for cancer immunization.

Tamoxifen chemoprevention of a hormone-independent tumor in the proto-neu transgenic mice model.

We evaluated the effect of tamoxifen (TAM) on tumor development in proto-neu transgenic mice that spontaneously develop mammary carcinomas overexpressing the neu protein. These mammary carcinomas are hormone independent because superimposable growth of transplants was observed in females and males. Virgin transgenic mice treated with TAM from 24 weeks of age, ie., when subclinical mammary tumors are already present, showed a slightly accelerated tumor development. In contrast, transgenic mice treated with TAM starting at 12 weeks of age, when subclinical tumors are not yet present, showed a 50% reduction of tumor incidence. Light microscopy analysis of the mammary gland of these mice revealed an apparently normal ductal branching but a complete regression of the acini. In conclusion, TAM can prevent the occurrence of hormone-independent breast carcinoma if given early enough to inhibit normal cells.

The 67 kDa laminin receptor increases tumor aggressiveness by remodeling laminin-1.

The association between expression of the 67 kDa laminin receptor (67LR) and tumor aggressiveness has been convincingly demonstrated although the exact function of this molecule in the metastatic process has remained unclear. In this study, we tested whether the laminin-1, upon interaction with 67LR, promotes tumor cell aggressiveness; the investigation was based on: (i) the previous demonstration that soluble 67LR, as well as a 20-amino-acid peptide corresponding to the 67LR laminin binding site, changes the conformation of laminin upon interaction with this adhesion molecule and (ii) the known relevance of microenvironment remodeling by the tumor, leading to structural modification of extracellular matrix components in tumor progression. MDAMB231 breast carcinoma cells plated on peptide G-treated laminin-1 exhibited a polygonal array of actin filament bundles compared with cells seeded on native laminin-1 which presented the actin bundles organized as multiple cables parallel to margins. Furthermore, in cells seeded on peptide G-treated laminin-1, 67LR was distinct from the alpha6 integrin subunit in filopodia protrusions in addition to colocalizing with this integrin in focal adhesion plaques as it occurs when cells are plated on native laminin-1. In addition to differences in tumor cell adhesion and migration found in cells exposed to peptide G-treated vs native laminin-1, breast carcinoma cells seeded on modified laminin-1 showed a 6-fold increase in invasion capability compared with cells seeded on unmodified laminin-1. Alterations in actin organization as well as adhesion, migration and especially invasion observed in MDAMB231 cells in the presence of peptide G-treated laminin-1 were even found in MDAMB231 cells that, after selection for 67LR high expression, were seeded on native laminin-1. As the 67LR shedding is proportional to its expression level, these findings indicate a role for 67LR in changing laminin structure. Expression analysis of 97 genes encoding proteins that mediate cell matrix interactions, revealed significant differences between cells exposed to modified vs unmodified laminin-1 in 19 genes, 17 of which--including those encoding alpha3 integrin, extracellular matrix protein 1, proteolytic enzymes (such as MT1-MMP, stromelysin-3 and cathepsin L) and their inhibitors--were up-modulated in cells treated with modified laminin-1. Zymogram analysis clearly indicated a significant increase in the activity of the gelatinolytic enzyme MMP-2 in the culture supernatant from cells exposed to modified laminin-1, without an increase in mRNA abundance as observed in microarray analysis. Invasiveness of tumor cells conditioned by modified laminin-1, evaluated as the capability to cross Matrigel basement, was significantly more inhibited by MMPinhibitor TIMP-2 than invasiveness induced by native laminin-1. Taken together, our findings indicate that the role of 67LR in tumor aggressiveness rests in its ability to modify laminin-1 thereby activating proteolytic enzymes that promote tumor cell invasion through extracellular matrix degradation.

Lymphoid infiltration as a prognostic variable for early-onset breast carcinomas.

Infiltration by lymphoid cells is a common feature of many human tumors, including breast carcinomas, and the degree of infiltration has been suggested to be a measure of the host immune response. Our analyses in a series of 1919 cases of primary ductal and lobular infiltrating breast carcinomas from women with a long-term follow-up revealed: (a) a 16-17% frequency of infiltrated tumors independent of the patient's age at diagnosis; and (b) a strong positive correlation between survival rates and the presence of lymphocytes at the tumor site in patients less than 40 years of age (P = 0.0002) but no association with prognosis in patients 40 years of age or older. Multivariate analysis indicated that lymphoid infiltration is independent of other conventional prognostic factors such as nodal status and tumor size in predicting survival. Thus, a possible immune response against the tumor seems to be relevant only in women with early-onset tumors. Because the immune system is functionally maximum in younger years, declining with age, this finding might reflect a difference in the efficiency of the immune system. Alternatively, the biology of these tumors might differ, leading to a difference in immuno-genicity.

Evaluation of the balance between angiogenic and antiangiogenic circulating factors in patients with breast and gastrointestinal cancers.

Angiogenesis is a critical determinant of tumor growth. Tumor cells produce or induce angiogenic molecules that act specifically on endothelial cells (ECs) but also release angiostatic molecules. Thus, tumor angiogenesis represents a net balance between positive and negative regulators of neovascularization. Sera from patients with breast or gastrointestinal cancers were evaluated for their capacity to selectively modulate the proliferation of human umbilical vein ECs; sera from 15 of 78 (19%) breast cancer patients and 8 of 53 (15%) gastrointestinal cancer patients induced human umbilical vein EC growth, whereas sera from 4 of 78 (5%) breast cancer patients and 1 of 53 (2%) gastrointestinal cancer patients inhibited EC proliferation. Growth-stimulatory sera were significantly more frequent among postmenopausal (14 of 53) than premenopausal (1 of 25) breast cancer patients; inhibitory activity was observed in 3 of 25 premenopausal patients versus 1 of 53 postmenopausal individuals. The half-life of serum-stimulating and -inhibiting factors seemed to differ, because stimulatory activity but not inhibitory activity was decreased at 5 days after surgery. The levels of vascular endothelial growth factor were elevated in about 45% of patients with growth-stimulatory sera, whereas the serum inhibition of EC growth was found to be due, at least in part, to high levels of soluble thrombospondin.

Nerve growth factor controls proliferation and progression of human prolactinoma cell lines through an autocrine mechanism.

Two different human prolactinoma phenotypes (responders and nonresponders), which are distinguished by different tumorigenic potential and different responsiveness to dopaminergic therapy, have recently been identified. Responders show low proliferation rate, low tumorigenic potential, and expression of D-2 receptors for dopamine (DA), while nonresponders are characterized by high proliferation rate, high tumorigenic potential, and lack of expression of DA D-2 receptors. In this study it has been shown that both gp140trk and gp75 components of nerve growth factor (NGF) receptor are expressed in responder prolactinoma cell lines. High levels of both NGF gene transcript and protein were also found in responders, and biologically active NGF was detectable in the media conditioned by these cells. Ablation of NGF production in responder cells by hybridization arrest of translation through NGF antisense oligonucleotides resulted in: 1) loss of secreted NGF; 2) loss of expression of gp75; 3) loss of expression of DA D-2 receptors; and 4) a remarkable increase in the cell proliferation rate. These results thus suggest that a NGF-mediated autocrine loop essential to control cell proliferation and to preserve some phenotypical characteristics of mammotroph cells is present in responder prolactinoma cell lines. Analysis of nonresponders showed that these cells express gp140trk but no detectable levels of gp75. In addition, no NGF mRNA or protein was detectable in nonresponders. Exposure of these cells to NGF resulted in the permanent expression of NGF mRNA and in the production and secretion of NGF protein, thus establishing the same NGF-mediated autocrine loop present in responders. As a result, it has been shown that nonresponder cells treated with NGF acquire and maintain most of the phenotypic characteristics of normal mammotroph cells. In conclusion, the present work reports that a NGF-mediated autocrine loop with an inhibitory role in the control of cell proliferation and tumor progression is active in the more differentiated DA-sensitive prolactinoma cell lines and is lost in the most malignant prolactinoma cells refractory to the dopaminergic therapy. Alterations in the expression of this autocrine loop thus may lead to cell transformation and tumor progression.

Generation of CD28- cells from long-term-stimulated CD8+CD28+ T cells: a possible mechanism accounting for the increased number of CD8+CD28- T cells in HIV-1-infected patients.

According to CD28 molecule expression, CD8+ T cells can be classed as CD28bright, CD28dim, and CD28-. The CD28dim T cells were found to derive from mitogenic stimulated CD28-T cells but also from CD28bright T cells through a mechanism of CD28 down-modulation. Moreover, after prolonged in vitro interleukin-2 stimulation, clonal CD28bright, cells showed a CD28dim expression before further evolution to a stable CD28-phenotype. This loss was concomitant with the disappearance of CD28 mRNA. A study of the cytokine production pattern revealed that CD28dim and CD28- T cell clones produced similar levels of type 1 and type 2 cytokines, which differed from those produced by the CD28bright T cell clones. A high percentage of CD28dim and CD28- cells, with similarities in their cytokine production pattern, were found in the blood samples of HIV-infected patients, as compared to healthy donors. The CD28 down-modulation may account for the increased number of CD8+CD28- T cells in HIV-infected patients.

Innate immunity in breast carcinoma.

The innate immune response, which depends on so-called pattern-recognition receptors (PRRs) is an evolutionarily old immune response able to elicit a defensive response against a vast array of pathogens. The purpose of this review is to revisit the role of innate immunity in breast carcinoma from the oldest therapeutic approach using bacillus Calmette-Guerin to the recent findings on the manipulation of the PRR pathways with unmethylated cytosine-guanosine dinucleotides (CpG motifs). Encouraging results have been obtained in prevention and local treatment of murine mammary tumors using tumor cells engineered to express stably mycobacterial antigens or directly using CpG-containing oligonucleotides. The experimental findings raise the possibility of successful anti-tumor management through stimulation of innate immunity in women at high risk of developing breast cancer and in breast cancer patients with reasonable immunological performance and low tumor load.

Nerve growth factor directs differentiation of the bipotential cell line GH-3 into the mammotroph phenotype.

GH-3 is an established cell line which, for the production of both PRL and GH, may be related to the bipotential somatomammotroph from which both somatotroph and mammotroph cells derive. In the present study we first report that GH-3 cells express both the gp140trk and the gp75 components of the nerve growth factor (NGF) receptor and that NGF dictates a nonneuronal type of differentiation of this cell line of ectodermal origin. After exposure to NGF, GH-3 cells markedly decreased their proliferation rate. This effect, which was maximal (50% inhibition) 3 days after beginning the treatment and was maintained during the following days of exposure, was paralleled by a change in the hormone production. The secretion of PRL was increased 6-fold, but that of GH was remarkably inhibited. Moreover, GH-3 cells expressed the mammotroph-specific D-2 receptor protein in response to NGF, as shown by binding with the D-2 receptor ligand N-(p-aminophenetyl)spiperone coupled to fluorescein. The present data thus show that NGF induces the differentiation of GH-3 cells into one of their physiological counterparts, the mammotroph cell, and together with the finding that NGF receptors are expressed in the anterior pituitary suggest a physiological role for the neurotrophic factor in pituitary ontogenesis.

A monoclonal antibody to the NH2-terminal region of human interferon-gamma inhibits its antiproliferative activity without affecting its internalization.

MAb IGMB-15, an anti-hIFN-gamma MAb, neutralizes the antiproliferative activity of hIFN-gamma without affecting that of hIFN-alpha or hIFN-beta. The neutralizing capacity of MAb IGMB-15 is wide: it has been assessed on cell lines whose origin and sensitivity to hIFN-gamma differ. The binding of hIFN-gamma to its receptor and its subsequent internalization into the target cell were not influenced by the antibody. MAb IGMB-15 has been found to interact with hIFN-gamma in solution but not when the lymphokine was associated with its cell surface receptor, showing that the growth of certain cell lines can be inhibited at the cell membrane level. This finding is consistent with the existence of an accessory factor responsible for the antiproliferative activity of hIFN-gamma.

Combination of a CpG-oligodeoxynucleotide and a topoisomerase I inhibitor in the therapy of human tumour xenografts.

The study was conducted to investigate the effects of a novel therapeutic approach, i.e. the combination of chemotherapy and immunotherapy, against a human prostate carcinoma xenograft. A topoisomerase I inhibitor, topotecan, and CpG-containing oligodeoxynucleotides (CpG-ODN) were combined. Athymic mice bearing the PC-3 human prostate carcinoma were treated with the maximum tolerated dose (MTD) of topotecan (3 weekly treatments) and with repeated treatments of CpG-ODN (40 and 20 microg/mouse); tumour growth and lethal toxicity were monitored. Topotecan effect on CpG-ODN-induced production of interleukin (IL) 12, interferon (IFN)-gamma and tumour necrosis factor-alpha was also assessed. Since topotecan pretreatment differentially influenced CpG-ODN-induced production of IL-12 and IFN-gamma, the antitumour effects of the two therapies were investigated in a sequential (full topotecan regimen followed by CpG-ODN) or in an alternating sequence (starting with CpG-ODN). Topotecan inhibited PC-3 tumour growth, inducing 95% tumour volume inhibition. All combined treatments resulted in a significant delay in tumour growth, compared to the effects in topotecan-treated mice (P<0.01, by analysis of tumour growth curves). The combination regimens were well tolerated, except for the alternating sequence of 40 microg CpG-ODN and topotecan, which resulted in three out of eight toxic deaths. This alternating sequence was highly toxic even when another cytotoxic drug (doxorubicin) was used in healthy mice. In conclusion, the combination of topotecan and CpG-ODN increased antitumour effects over chemotherapy alone in the growth of a human prostate carcinoma xenograft. Administration sequence was critical to the combination toxicity: the complete regimen of the cytotoxic drug followed by repeated administrations of the immunomodulator seemed the most promising for further investigations.