Superantigens but not mitogens are capable of inducing upregulation of E-selectin ligands on human T lymphocytes

Abstract Bacterial infections can exacerbate immune mediated dermatoses, possibly via Superantigens produced by these bacteria. Therefore, we asked whether Superantigens induce the expression of adhesion molecules which may then facilitate invasion of highly activated T cells into different organs. The influence of exfoliative toxin (ET) and toxic shock syndrome toxin-1 (TSST-1) stimulation on the expression of a broad panel of adhesion and coslimulatory molecules was investigated by flow cytometry. We found that only the E-selectin ligands cutaneous lymphocyte-associated antigen (CLA) and sialylated Lewis^x (CD15_s) are significantly upregulated by these Superantigens but not by mitogen stimulation. In contrast, the mucosal lymphocyte-associated antigen (MLA) recognized by the monoclonal antibody Ber-Act8 was.not differentially induced by mitogen or superantigen stimulation. Therefore, T lymphocyte stimulation by bacterial Superantigens might directly influence their skin homing capacity. Furthermore, the superantigen-driven induction of CD15_s - an adhesion molecule which is absent or only weakly expressed by resting or mitogen stimulated T cells may indicate a role of this antigen for T cell skin homing. An additional adhesion pathway via E-selectin may thus be available to lymphocytes, comparable to granulocyles which constitutively express CD15_s.     

Adhesion molecules in atopic dermatitis: patch tests elicited by house dust mite

Different T-helper subsets, which are characterized by the secretion of distinct cytokines (Th1, Th2), have been found in house dust mite-exposed skin of sensitized individuals and in nickel-specific T lymphocytes from nickel contact allergic and non-allergic individuals. In order to evaluate the role which adhesion molecules may play in the homing of different T-cell subsets into allergen-exposed skin of atopic and normal individuals, we compared the expression pattern of adhesion molecules in patch test reactions to house dust mite antigen (D.pt.), nickel sulfate (Ni) and the irritant anthralin. Biopsies were taken al various time points after application of these agents and studied by immuncytochemistry. To exclude an endogenous difference in adhesion molecule expression in atopic and non-atopic skin, sequential biopsies from Ni patch tests of 2 normal individuals were also included in this study. The expression of E-selection. P-selection. CD31, VCAM-1 and ICAM-1 on endothelial cells and other cells in the skin was quantified by microscopic evaluation. Skin homing T cells were also quantified using antibodies toCD3, CD4, CDS, UCHL-I, L-selection and the cutaneous lymphocyte antigen (CLA). Independent of the eliciting substance, all lesions showed an up regulation of all adhesion molecules tested, with the exception of CD62. The appearance of E-selection and an increase in ICAM-1 and VCA M-1 expression were first observed at 12 h after application of the various agents. In parallel, the number of CLA+ and L-selection+ lymphocytes increased steadily. No principle differences could be established between the various types of skin reactions in atopic individuals, nor did the skin of patients with AD differ from normal controls. Our results provide evidence that differential expression of adhesion molecules does not play a major part in observed differential homing of Th1 and Th2-cell subsets into patch test sites provoked by house dust mite and nickel sulfate in atopic and non-atopic individuals.     

PHARMACOLOGIC MODULATION BY CETIRIZINE OF SOME ADHESION MOLECULES EXPRESSION IN PSORIATIC SKIN LESIONS

Background. Adhesion molecules play a major role in the pathogenesis of inflammatory skin diseases by regulating lymphocyte trafficking and homing in an inflamed area. Methods. The expression of the lymphocyte function-associated antigen-1 (LFA-1) and of its ligand, the intercellular adhesion molecule-1 (ICAM-I) has been studied in psoriatic skin lesions of 10 patients with guttate, nummular, and palmoplantar psoriasis. In addition, the peculiar immunophenotype of infiltrating cells (CD3, CD4, CD8, CD25) and their correlation with HLA-DR expression before and after treatment with oral cetirizine, a highly selective, third generation H_1-receptor antagonist has been examined using the labeled avidin biotin (LAB) system. Results. Cetirizine treatment modulated in vivo the expression of adhesion molecules LFA-I/ICAM-1 as shown in all cases by decreased levels of their expression on keratinocytes and on dermal endothelial cells (P < 0.001). The expression of HLA-DR on keratinocytes and endothelial cells was also inhibited after treatment. The numbers of infiltrating CD3–, CD4–, CD8–positive cells were reduced, whereas there was no significant modification of CD25–positive cells within the epidermis and the dermis. Conclusion. This open clinical trial suggests that cetirizine could be effective in treating psoriasis: (1) for its symptomatic control on itching; (2) for its immunopharmacologic modulation of leukocyte integrins and on the immunophenotype pattern of infiltrating and resident cells, and (3) for contributing to the clearing of the lesions clinically.     

The development of a Ki-1-positive large cell non-Hodgkin's lymphoma in pagetoid reticulosis

A case of the disseminated variant of pagetoid reticulosis is described which progressed after many years of disease to a large cell anaplastic (Ki-1) lymphoma of T-cell type. The lymphoma cells showed abnormal cellular DNA and a high proliferative rate, as revealed by immunophenotypic examination and single-cell DNA measurements. The cells were positive for activation associated antigens and expressed a T-helper/inducer phenotype. A similar phenotype was expressed by the neoplastic cells in the co-existing lesions of pagetoid reticulosis. These findings support the view that pagetoid reticulosis is a variant of the cutaneous T-cell lymphomas which originates from activated lymphoid cells and which may on occasion progress to a potentionally more aggressive lymphoid malignancy.     

Pagetoid reticulosis (Woringer-Kolopp disease): histiocyte marker (lysozyme) study and ultrastructural observations

A case of pagetoid reticulosis is presented. Histopathology showed infiltration of the epidermis by mononuclear cells. Twenty percent of the mononuclear cells showed the presence of lysozyme indicating a histiocytic origin. Electron microscopy confirmed the presence of lymphocytes and histiocytes but these cells were outnumbered by Sézary cells. The presence of large numbers of Sézary cells indicates that pagetoid reticulosis is a cutaneous T-cell lymphoma closely related to mycosis fungoides.     

Pagetoid Reticulosis and Solitary Mycosis Fungoides

The two cases reported in this paper allow the differentiation pagetoid reticulosis from solitary lesions of mycosis on the basis of clinical, histological ultrustructural immunohistochemical and enzyme histochemical studies. The first patient presented with a slowly growing asymptomatic scaly plaque of 30 years duration Histology of the lesion was typical of pagetoid reticulosis. Immunohistochemical studies showed that the abnormal cells present in the cutaneous infiltrate were not T lymphocytes and ultrastructural studies suggested these were of histocytic origin. The second patient presented with a poikilodermatous lesion present for 14 years with the histology of plaque stage mycosis fungoides. Enzyme and immunohistochemistry showed the predominant T cell nature of the cutaneous infiltrate and electron microscopy showed numerous mycosis fungoides cells. In contrast to generalized forms of mycosis fungoides, no peripheral blood monocyte chemotactic defect was found in either case. Both lesions responded to radiotherapy.     

Immunological effects of stress in psoriasis

Background Psychological stress causes phenotypic changes in circulating lymphocytes and is regarded as an important trigger of the Th1‐polarized inflammatory skin disease psoriasis. Objective To study the effects of psychological stress on immunological parameters, i.e. membrane molecules relevant to the pathophysiology of psoriasis, especially cutaneous lymphocyte‐associated antigens (CLA) involved in T and natural killer (NK) cells homing in on the skin. Methods The severity of psoriasis was assessed in patients using the Psoriasis Area and Severity Index. Patients with psoriasis (n = 15) and healthy volunteers (n = 15) were exposed to brief psychological stress in the laboratory. In vitro analyses were conducted 1 h before, immediately following and 1 h after stress exposure. Peripheral T‐ and NK‐cell subsets including CD8+ T lymphocytes, CLA+ lymphocytes and lymphocyte function‐associated antigen type 1 (LFA‐1)+ lymphocytes were analysed by flow cytometry. Results We found a significant stress‐induced increase of CD3+ T lymphocytes in patients with psoriasis only. Analyses of T‐cell subsets revealed that this increase was observable for cytotoxic CD8+ T lymphocytes and CLA+ CD3+ lymphocytes. The total number of circulating NK cells (CD16+, CD56+) increased immediately after stress in both groups whereas only patients with psoriasis showed a significant increase in CLA+ NK cells. Conclusions A higher stress‐induced increase of CLA+ T and CLA+ NK cells in the circulation of patients with psoriasis might point to an increased ability of T and NK cells in the presence of psoriasis to home in on the skin during mental stress. Further studies are needed to verify these relationships in more detail and to investigate the time point at which these cells accumulate within lesional skin, and whether or not psychotherapy improves the quality of life of patients with psoriasis and influences stress‐dependent parameters.     

Skin-homing T lymphocytes: detection of cutaneous lymphocyteassociated antigen (CLA) by HECA-452 in normal human skin

The immigration of circulating T cells into specific tissues is directed by the interaction between adhesion molecules on lymphocyte subpopulations and their ligands on vascular endothelium. Of these, endothelial leucocyte adhesion molecule (ELAM-1), weakly expressed in normal human skin (NHS), seems to be the counter-structure for cutaneous lymphocyte-associated antigen (CLA). CLA is a 200 kDa cell-surface glycoprotein of which the sugar moieties sialyl Le(a) and sialyl Le(x) are the possible epitopes recognized by the monoclonal antibody HECA-452. HECA-452 was originally described as a marker for lymphoid organ high endothelial cells, but 16% of peripheral-blood-derived T cells react with this antibody. We studied the expression of CLA on the cellular constituents of the skin immune system (SIS). By applying immunohistochemical double staining, 41% of CD3+ T cells, 44% of CD4+ T cells and 31% of CD8+ T cells were found to express CLA. Keratinocytes, CD1a+ Langerhans cells (LC) and endothelial cells did not express HECA-452 in significant numbers in NHS. Monocytes were found to express HECA-452 in 14% of CD68+ cells. CLA expression was present on a relatively low percentage of T cells and subsets localized distant from NHS vessels, suggesting loss of the molecule during further migration after transendothelial passage. However, intraepidermal T cells expressed CLA in similar percentages to T cells localized directly perivascularly. Our findings support the notion that CLA expression by T cells is associated with their homing into cutaneous structures.     

Efalizumab therapy for atopic dermatitis causes marked increases in circulating effector memory CD4+ T cells that express cutaneous lymphocyte antigen

Efalizumab is an mAb directed against CD11a, a molecule involved in T-cell activation and extravasation from blood into tissue. Ten patients with severe atopic dermatitis were treated with efalizumab for 84 days, and peripheral blood mononuclear cells were analyzed for expression of activation and adhesion markers. Efalizumab treatment led to decreases in CD11a mean fluorescence intensity (MFI) on naive, central memory, and effector memory CD4+ and CD8+ T cell subsets. MFI for CD18 was decreased in both CD4+ and CD8+ T cells. Percentages of cells positive for cutaneous lymphocyte antigen (CLA) were increased fourfold in all CD4+ and CD8+ T cell subsets. Increases in the percentages of CD4+ and CD8+ T cells expressing beta7 and CD49d were also observed. No significant changes were observed in the percentages of CD4+ and CD8+ T cells that produced either IFN-gamma or IL-4. In summary, efalizumab treatment resulted in (i) decreases in CD11a and CD18 expression in all circulating T-cell subsets and (ii) increases in the percentages of blood T cells expressing tissue homing markers (CLA, beta7, CD49d). These data suggest that blockade of T-cell extravasation into tissue is the major pathway by which efalizumab leads to improvement in cutaneous inflammation.     

Pagetoid reticulosis of the Ketron-Goodman type

The case of a 79-year-old woman with widespread pagetoid reticulosis (Ketron-Goodman type) is presented. The nature of the epidermal cellular infiltrate in pagetoid reticulosis of the Ketron-Goodman type was examined by immunohistochemical methods and by electron microscopy. On ultrastructural examination the infiltrating cells proved to be medium-sized and large Sézary-type and blastoid cells. Immunohistochemically, the epidermal infiltrate was composed of approximately 80% immature T cells (OKT 16) and 30% proliferating cells (Ki 67). Mature T cells (CD 5) were found to account for only 15-30% of all cells in the infiltrate. These results suggest that pagetoid reticulosis (Ketron-Goodman type) is a malignant T-cell lymphoma, which is characterized, in addition to the epidermotropic behavior common to all T-cell lymphomas, by innidiation and in-situ spread within the epidermis.     

银屑病患者皮损及非皮损部位粘附分子免疫组化研究

目的　为了更好地了解浸润 T淋巴细胞和内皮细胞粘附分子的原位表达在银屑病皮损中的相互关系。方法　采用免疫组化染色方法研究银屑病皮损部位和非皮损部位的浸润 T淋巴细胞亚群和内皮细胞粘附分子 (ICAM- 1,EL AM- 1,VCAM- 1)的原位表达情况。结果　银屑病患者皮损部位 T细胞亚群和内皮细胞粘附分子的原位表达均显著高于非皮损部位 ,而且浸润 T淋巴细胞亚群的细胞密度和内皮细胞粘附分子的原位表达程度呈显著正相关。与正常人相比 ,银屑病非皮损部位和经外用皮质类固醇激素治疗后外观正常的皮肤内皮粘附分子仍呈上调表达。结论　银屑病患者皮肤真皮血管内皮细胞粘附分子的异常上调表达是造成银屑病复发的原因之一。     

Monocytic leukemia. Clinically appearing as 'malignant reticulosis of the skin'.

Enzyme cytochemical (hydrolytic enzymes for cell differentiation), immunocytologic (B and T lymphocyte differentiation), and electron microscopic studies of skin infiltrates facilitate the proper diagnosis of myelomonocytic and lymphoreticular proliferations. With use of these methods, the original clinical diagnosis of malignant reticulosis of the skin was corrected to monocytic leukemia in a 65-year-old woman. Because primary involvement of the skin preceded monocytosis of the blood, it was concluded that the cutaneous infiltrates in our patient resulted from proliferation of tumor cells in the skin rather than from homing of the cells to, or settling of the cells in, the skin.     

Adhesion molecules in lesions of American cutaneous leishmaniasis

Abstract Accessory signals, which include adhesion molecules, MHC-II molecules and cytokines. are necessary to foster the interaction between memory T cells and epidermal cells, that is required to promote cutaneous inflammatory responses. American cutaneous leishmaniasis (ACL) is characterized by a spectrum of immunological manifestations, and is a prototype disease for the study of regulatory mechanisms involved in immune protection against protozoal infection. In the present study, we show that diffuse cutaneous leishmaniasis (DCL) epidermis contains keratinocytes that do not express ICAM-I and HLA-DR molecules. Langerhans cells (LC) are within normal values or somewhat lower, and a very few cells expressing the HB15 molecule a new described member of the Ig superfamily are found in such lesions. Mucocutaneous leishmaniasis (MCL) epithelium shows an increased expression of ICAM-1 and HLA-DR molecules, few HBI5⁺ cells, and an absence of epithelial LC. Localized cutaneous leishmaniasis (LCL) epidermis displays ICAM-⁺ keratinocytes organized in patches, a uniform expression of HLA-DR, hyper-plasia of LC, and numerous HBI5⁺ cells. In all forms of the disease, infiltrating T cells express more LFA-1β than LFA-1α, but LFA-1β⁺ cells are more abundant in LCL granulomas. In contrast, there are more LFA-lα⁺ cells in DCL and MCL than in LCL granulomas. LCL lesions also show the highest numbers of HB15⁺ cells within the granu-loma. These results indicate the importance of adhesion molecules in ACL lesions, and open new possibilities for therapeutic schemes oriented towards the control of cell migration.     

1,24‐Dihydroxyvitamin D3 (tacalcitol) prevents skin T‐cell infiltration

Background 1,24‐Dihydroxyvitamin D₃ (tacalcitol), a vitamin D₃ compound, has been used to treat T cell‐mediated inflammatory skin diseases such as psoriasis, prurigo and vitiligo. The best‐known mechanism of action of this compound is inhibition of the abnormal proliferation of keratinocytes and subsequent maturation; however, its effects on skin T‐cell recruitment have not yet been evaluated. Cutaneous lymphocyte‐associated antigen (CLA), a surface glycoprotein expressed on T cells, plays a critical role in skin T‐cell infiltration. We recently reported that 1,25‐dihydroxyvitamin D₃ inhibits skin infiltration of CD4+ T cells by suppressing CLA expression on T cells. Objectives In this study, we investigated the effect of tacalcitol on CLA epitope decoration and on the levels of gut or lymph node homing receptor expression in human T cells. Methods We cultured human T cells with tacalcitol and analysed the effect on CLA expression and skin‐homing ability, and evaluated glycosyltransferase mRNAs. We also performed an in vivo study using an antigen‐dependent delayed‐type hypersensitivity (DTH) mouse model and investigated the effect of tacalcitol on skin‐infiltrating CD4+ T cells. Results Tacalcitol downregulated the expression of CLA and, in parallel, the E‐ and P‐selectin ligand function; however, it exerted no effect on other homing receptors. Subcutaneously and intraperitoneally administered tacalcitol downregulated skin infiltration of effector CD4+ T cells in an in vivo DTH mouse model. Conclusions These findings suggest that tacalcitol reduces skin inflammation by partially downregulating CLA expression levels.     

Disseminated pagetoid reticulosis: plaques and tumoral lesions occurring simultaneously in the same patient

Disseminated pagetoid reticulosis is considered to have a poor prognosis. Some authors regard this disease as an epidermotropic type of mycosis fungoides rather than a distinct entity. We report the case of an 83-year-old woman with disseminated pagetoid reticulosis who subsequently developed tumoral lesions and died soon after the appearance of these tumours. Our case is in agreement with findings that disseminated pagetoid reticulosis has an aggressive clinical behaviour.     

Photochemotherapy for systemic sclerosis: effect on clinical and molecular markers

Background. The cutaneous changes seen in systemic sclerosis (SSc) can result in considerable patient morbidity. Aim. We previously reported on the beneficial effect of psoralen ultraviolet A (PUVA) phototherapy in 13 patients with morphoea. We now report the findings of a study in which patients with SSc were treated with PUVA. Methods. Twelve patients with SSc were treated with PUVA phototherapy. The effect on cutaneous disease activity was assessed using the modified Rodnan score, and the effect on serological and immunohistochemical growth factors and adhesion molecules was also measured. Results. The median Rodnan score at baseline was 24.5 [interquartile range (IQR) 18.5–26.0]. The median number of treatments with PUVA was 24 exposures (IQR 20–26) with a median cumulative exposure of 68.3 J/cm² (IQR 28.6–139.8). Of the 12 patients, 11 responded well to phototherapy with a mean change in Rodnan score of 6.58 (36.98%) (P < 0.01, Wilcoxon signed ranks test). After treatment with PUVA there was a significant increase in circulating tumour necrosis factor‐α levels in 8/12 patients (P = 0.03). In 7/12 patients there was an increase in E‐selectin and vascular cell adhesion molecule, although this was not significant. Conclusions. PUVA treatment is associated with a significant improvement in cutaneous symptoms in patients with SSc as measured by the Rodnan score (P < 0.01). Specific lymphocyte markers, adhesion molecules and cytokines are also affected by this treatment, helping to clarify further the mechanism of action of PUVA treatment and our understanding of the primary pathological process.     

Función efectora de linfocitos T CLA+ sobre queratinocitos autólogos en psoriasis

BackgroundCutaneous lymphocyte antigen (CLA) is expressed by a subgroup of memory T cells that exhibit skin homing and are implicated in cutaneous T-cell-mediated diseases.Material and methodsExpression of genes associated with psoriasis was analyzed in keratinocytes taken from patients and healthy individuals and cultured under different conditions, including activation using supernatants from CLA⁺ T lymphocytes activated with anti-CD3 and anti-CD28 antibodies.ResultsKeratinocytes from psoriasis patients activated by CLA⁺ T lymphocytes expressed higher levels of interferon-inducible protein 10, HLA-DR, intercellular cell adhesion molecule 1, and inducible nitric oxide synthase.ConclusionsOur results suggest that we have developed an in vitro model that will allow analysis of the effector role of CLA⁺ T lymphocytes on keratinocytes in psoriasis. This model may allow the identification of genes involved in the pathology of psoriasis through induction by CLA⁺ T lymphocytes.     

CD4/CD8 double negative pagetoid reticulosis: a case report and literature review

Pagetoid reticulosis is an indolent primary cutaneous T‐cell lymphoma. It typically presents as a solitary and slowly growing patch or plaque on the extremity, histologically characterized by an acanthotic epidermis infiltrated with atypical lymphocytes. Here, we present histological, immunophenotypical and molecular findings of a 29‐year‐old Jamaican man with bilateral wrist plaques. Histology showed marked acanthosis, hyperkeratosis and an intraepidermal infiltration consisting of large atypical lymphocytes. Immunohistochemical stains showed CD3 and CD5 positive T cells with significant loss of CD7, double negative CD4 and CD8 and strong positive CD30. Molecular analysis showed a monoclonal T‐cell receptor (TCR) gamma gene rearrangement. Review of the literature confirms that the immunophenotype of pagetoid reticulosis is variable with decreasing frequency of CD8+ cytotoxic/suppressor T cell, CD4+ helper T cell and least commonly CD4/CD8 double negative phenotypes. Although CD4/CD8 double negative phenotype appears to be associated with higher proliferation index, it does not appear to confer prognostic significance. Mourtzinos N, Puri PK, Guanghua W, Liu M‐L. CD4/CD8 double negative pagetoid reticulosis: a case report and literature review.     

Reduction of skin-homing cytotoxic T cells (CD8+ -CLA+) in patients with vitiligo

Background: Vitiligo is a frequently acquired, hereditary disease, characterized by achromic macules due to the absence of melanocytes. In contrast with earlier studies, in which the main pathogenic role was attributed to anti‐melanocyte antibodies, recent papers have emphasized a role for CD8⁺ cytotoxic T lymphocytes in melanocyte destruction. Fifteen percent of peripheral T cell express cutaneous lymphocyte‐associated antigen (CLA), responsible for skin‐homing T cell. Phototherapy is used to treat patients with generalized vitiligo and it has been shown to interfere with CLA⁺ T cells in other skin diseases. Objective: To describe peripheral blood T cell subpopulations' frequency and ability to express the skin‐homing molecule (CLA) in patients with non‐segmental vitiligo, before and after photochemotherapy (PUVA). Patients and Methods: Twenty‐two patients with generalized and active spreading vitiligo were submitted to 30 PUVA‐8MOP sessions. Lymphocyte immunophenotyping was performed by flow cytometry using anti‐CD3, anti‐CD8 and anti‐CLA monoclonal antibodies. Fifteen healthy volunteers, sex‐ and age‐matched, were included as a control group. Results: CD8⁺–CLA⁺ T cells were significantly reduced in number in untreated vitiligo patients (P=0.008) when compared with control individuals, albeit with a more intense CLA expression (P=0.028). These findings were not altered after PUVA. No significant difference was noticed in CD4/CD8 ratios nor in CD4–CLA⁺ T cell numbers between vitiligo patients and controls, both before and after PUVA. Conclusions: CD8–CLA⁺ T cells are reduced in peripheral blood of patients with non‐segmental vitiligo. This finding may be related to the previously reported increase of CD8⁺ cells in both lesions and perilesional skin of these patients.     

Visualizing CD4 T‐cell migration into inflamed skin and its inhibition by CCR4/CCR10 blockades using in vivo imaging model

Background Chemokines are critical mediators of T‐cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo. Objectives To visualize directly T‐cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy. Materials and methods A mouse model of allergic contact dermatitis was used. T cells from oxazolone‐sensitized and ‐challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c‐SCID mice. The recipient mice were challenged with oxazolone and CD4 T‐cell homing into inflamed skin was visualized. Results T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T‐cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T‐cell migration and skin inflammation. Conclusions Our data indicate that these tissue‐selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T‐cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.