Triple Color Research Articles

Antigen-specific cytokine response to hepatitis C virus core epitopes in HIV/hepatitis C virus-coinfected patients.

Epidemiological data indicate that hepatitis C virus (HCV) infection runs a more rapid and severe course of disease in HIV-coinfected patients, probably because of an altered immune response. We investigated whether HCV-specific cytokine responses are affected by HIV coinfection. Using triple colour flow cytometry on peripheral blood lymphocytes after stimulation with the four major immunodominant HCV core T cell epitopes, CT1-CT4, we determined intracytoplasmic production of IFN-gamma, IL-2, IL-4, IL-10 and CD30 expression, a putative surrogate marker of type 2 cells. Fifteen patients with asymptomatic HIV/HCV coinfection (group A), 15 patients with chronic HCV infection (group B) and 10 HIV-infected patients without hepatitis C (group C) were included in the study. In group A, HCV antigens induced significantly higher IL-2 and IFN-gamma production than groups B and C (P < 0.05). Groups A and B showed a similar induction of CD30, which was significantly higher than in group C (P < 0.001). Remarkably, in group A HCV antigens induced IL-4 production in addition to IL-10 and IFN-gamma in the CD30 subset, whereas in groups B and C no IL-4 induction was observed in this T cell subset (P < 0.002). Our data suggest that asymptomatic HIV coinfection importantly alters the HCV-specific cytokine response towards a greater production of proinflammatory type 1 cytokines. Moreover, the antiviral activity of type 1 cytokines may be modified by an increased production of type 2 cytokines in the CD30 subset. The altered cytokine pattern may contribute to the adverse natural course of hepatitis C in HIV coinfection.

More worries about ultrasound

One of the best kept secrets on ultrasound is the huge increase in exposure unborn babies are getting from the newer, more powerful equipment. Obstetricians are so excited by the great pictures they get from the triple colour Doppler, they dont want to hear the bad news. You dont hear the scary stuff until you start talking to the physicists, and when they get worried, consumer groups start to listen.

Variation in gut-homing CD27-negative lymphocytes in inflammatory colon disease.

Prolonged antigenic stimulation results in lymphocyte shedding of CD27, a member of the tumour necrosis factor receptor (TNFR) family, and transformation to a stable phenotype capable of synthesizing interleukin-4 (IL-4). Co-expression of alpha4beta7 identifies those cells with gut-homing potential. We have investigated these cell populations in patients with inflammatory colonic disease. Circulating and lamina propria mononuclear cells were isolated from patients with Crohn's disease (CD), ulcerative colitis (UC), non-inflammatory bowel disease (non-IBD) colonic inflammation and healthy controls. Double and triple colour flow cytometry for CD3, CD4, CD27, alpha4beta7 and intracellular cytokines was performed. Circulating CD4+ CD27- populations were increased in patients with CD (8.8 +/- 0.8%, P < 0.001), UC (12.2 +/- 1.9%, P < 0.001) and non-IBD colitis (10.5 +/- 1.3%, P < 0.01) as compared with controls (6.1 +/- 0.5%). CD4+ CD27- alpha4beta7+ cells were increased in CD (P < 0.01). Lamina propria CD4+ CD27- populations were depressed significantly in CD (P < 0.05), UC (P < 0.02) and non-IBD colitis (P < 0.03). Mucosal CD4+ CD27- cells synthesized IL-4 in preference to interferon-gamma. Thus, colonic inflammation is associated with alterations in gut-tropic circulating and mucosal populations of differentiated memory T cells with the phenotype of predominantly IL-4-synthesizing cells.

Dose-response curves for X-ray induced interchanges and interarm intrachanges in human lymphocytes using arm-specific probes for chromosome 1

Frequencies of chromosomal aberrations were estimated in X-irradiated (0; 0.5; 1; 2; 3 and 4 Gy) human lymphocytes using arm-specific probes for chromosome 1 in combination with a pancentromeric probe in a triple colour FISH procedure. This allowed the construction of dose-response curves for both interchanges as well as interarm intrachanges. We found that 11.7–18.4% of aberrant chromosomes contained interarm intrachanges. Assuming a free interaction of randomly induced exchange breakpoints throughout the whole genome, interarm intrachanges occur about 8.7 times more frequently than expected. The close proximity of the two arms of a chromosome in the interphase nucleus appears to promote the formation of interarm intrachanges. Convincing evidence was obtained, by comparing the frequency of colour junctions that occur between 1p–1q and 1p–3q (the arms 1q and 3q are of similar size). With this approach, we observed 8.8 time more colour junctions between 1p–1q than between 1p–3q, illustrating the importance of proximity in the formation of chromosomal exchange aberrations. At doses higher than 2 Gy, a saturation of simple reciprocal aberrations, both for interchanges as well as for interarm intrachanges was observed. Furthermore, we found that symmetrical and assymmetrical simple interchanges as well as interarm intrachanges occur with equal frequencies in human chromosome 1.

A case with mosaic di-, tetra-, and octacentric ring Y chromosomes

A newborn female infant presented with abnormalities of the external genitalia including a 3 x 1 cm phallic structure, a perineal urethral opening, bifid scrotum, and a single urogenital opening. Peripheral blood karyotype was 45,X[81]/46,X,+r(Y)[19], however, there were no signs of Ullrich-Turner syndrome. High resolution G-banding as well as C- and Q-banding did not demonstrate any specific banding pattern or presence of heterochromatin on the ring. However, it was noticed that some of the rings were larger than others. FISH with a probe for Yq12 was negative in all metaphases studied. A Y-specific paint probe hybridized to the entire ring chromosome, confirming its origin. PCR analysis showed the presence of the SRY locus and of proximal Yq locus DYS271. Triple color FISH with probes for the Y centromere, DYZ5 (Yp), and all human telomeres showed the existence of different types of rings, some dicentric, some tetracentric, and some probably octacentric. Owing to the increased risk for gonadoblastoma, a surgical removal of the gonads was performed.

Familial four breakpoint complex chromosomal rearrangement as a cause of monosomy 9p22-->pter and trisomy 10p11.2-->pter and 11q21 analysed by dual and triple colour FISH.

A familial four breakpoint complex chromosomal rearrangement involving chromosomes 9, 10, and 11 was ascertained through a child with dysmorphic features, hypertrophic cardiomyopathy, and hypotonia. A cryptic insertion, invisible in...

Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry.

To obtain reference values of the level of expression of T cell antigens on normal lymphocyte subsets in order to disclose differences which could reflect their function or maturation stages, or both. Peripheral blood from 15 healthy donors was processed by flow cytometry with triple colour analysis. For each sample phycoerythrin (PE) conjugated CD2, CD4, CD5, CD8, and CD56 monoclonal antibodies were combined with Cy5-R-phycoerythrin (TC) conjugated CD3 and fluorescein isothiocyanate (FITC) conjugated CD7; CD2- and CD7-PE were also combined with CD3-TC and CD4-FITC. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values of the lymphocyte subsets identified by multiparametric flow cytometry into the number of antigen molecules per cell, measured as antibody binding capacity (ABC). CD4+ (helper/inducer) T cells exhibit a higher CD3 antigen expression compared with CD8+ (suppressor/ cytotoxic) T lymphocytes. Within the CD4+ T cells, the CD4+CD7- subset expressed a lower level of CD3 compared with CD4+CD7+ and CD8+CD7+ cells, and higher CD2 and CD5 expression than the main CD3+CD7+ subset. Major differences in antigen expression were also detected between CD3+ T cells and CD3-CD56+ natural killer (NK) cells: NK cells exhibited higher levels of CD7 and CD56 and lower levels of CD2 and CD5 than T cells. Significantly lower CD5 expression was also detected in the small CD5+ B lymphocyte subset compared with T cells. Quantitative flow cytometry with triple colour analysis may be used to detect antigen modulations in disease states and to increase the accuracy of diagnosis by comparison with findings in normal counterparts.

Retinoic Acid Upregulates Human Langerhans Cell Antigen Presentation and Surface Expression of HLA-DR and CD11c, a β2 Integrin Critically Involved in T-Cell Activation

Immunomodulatory effects of retinoids may be part of their anti-carcinogenic and anti-inflammatory properties. We studied the in vivo effects of retinoic acid (RA) on antigen-presenting activity of human epidermal Langerhans cells and on accessory cell activity of keratinocytes. Two skin sites from each volunteer were treated in vivo with 0.1% RA or vehicle, respectively, once a day for 4 d. RA-treated epidermal cell (RA-EC) alloantigen presentation to CD4+ T cells in each volunteer tested was consistently greater than that induced by vehicle EC. However, this increased antigen-presenting activity did not lead to autoreactive CD4+ T-lymphocyte proliferation. Elevated unfractionated epidermal antigen-presenting activity of RA-EC was not due to increased keratinocyte major histocompatibility complex (MHC) or intercellular adhesion molecule expression or to other keratinocyte accessory signaling, because incubation of CD1a-fluoroscence-activated cell sorter (FACS)-purified RA-EC inhibited alloantigen presentation, presumably through increased keratinocyte transforming growth factor-beta. By contrast, Langerhans cell function was upregulated; FACS-purified CD1a+ Langerhans cells derived from RA-EC displayed a markedly increased ability, relative to Langerhans cells from vehicle EC, to present alloantigen to T cells. Triple color flow-cytometric analysis of RA-EC and vehicle EC suspensions revealed that RA treatment did not modify the number of DR+ and CD1a+DR+EC, but did result in statistically significant increases in Langerhans cells expression of HLA-DR, CD11c, and CD1c. Another novel finding was that HLA-DR-dependent Langerhans cells antigen-presenting activity in both normal and RA-treated skin was completely blocked by anti-CD11c antibody. Thus, retinoid upregulation of antigen-presenting activity may be due to upregulation of Langerhans cell CD11c, as well as class II MHC. Upregulation of cutaneous immune responsiveness in human skin without autoreactivity has not (to our knowledge) been reported previously, and the Langerhans cell phenotypic and functional state achieved is distinct from previously reported states of Langerhans cell activation.

Human peripheral blood dendritic cell subsets. Isolation and characterization of precursor and mature antigen-presenting cells.

Dendritic cells (DC) are the major APC capable of stimulating resting T cells in human peripheral blood (PB). Recent evidence suggested that various subsets of DC and monocytes might circulate in human PB, but their exact phenotype and function had not been delineated. We have previously characterized a population of human PB DC precursors that express the myeloid marker CD33, but not the monocyte marker CD14. To identify and characterize further functional myeloid APC subsets, triple color FACS analysis and sorting was used. A CD33dimCD14dimCD16+ monocyte subset, with similar APC function but less efficient accessory function than CD14bright monocytes, was isolated. In addition to the CD33+CD14dimCD16- DC precursors, a smaller population (0.1 to 0.2% of PBMC) of CD33brightCD14dimCD16- cells with potent APC function was identified. This DC population expressed greater amounts of MHC class II, adhesion, and accessory molecules, and demonstrated a greater costimulatory capacity when freshly isolated than CD33dimCD14dim DC precursors, and therefore had the characteristics of mature, possibly tissue-derived DC. When freshly isolated, however, these DC did not express B7, and up-regulation of accessory function occurred after in vitro differentiation. These data demonstrate multiple circulating myeloid accessory and APC subsets in human PB. Phenotypic and functional differences suggest that they are at different stages of differentiation, and have specialized roles in Ag presentation in vivo. Furthermore, full functional DC differentiation, associated with B7 expression and the capacity to activate T cells maximally, is likely to occur only in specific physiologic circumstances.

Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets

Abstract The relative contribution of dermal-derived immunocompetent cells to the overall immunologic response in skin has been hampered by the lack of appropriate isolation techniques. In this report, we provide a purification schema that reliably yields highly purified populations of dermal dendritic cells (DDC). These cells are motile, express high levels of class II MHC antigens that decorate their cytoplasmic dendritic processes, and lack numerous B cell, T cell, and natural killer cell antigens. Using a broad panel of 45 different antibodies, an extensive phenotypic analysis was completed, revealing distinctive profiles for subsets of DDC. Despite homogeneous light scatter profile and cytologic appearance, three subsets of DDC could be distinguished by phenotypic and functional criteria. All DDC, but not epidermal Langerhans cells, express factor XIIIa. By triple color cell staining the relative distribution of factor XIIIa positive DDC is as follows: subset 1, 65% to 70% of total DDC express neither CD1a nor CD14; subset 2, 15% to 20% of total DDC express CD1a but not CD14; and subset 3, 10% to 15% of total DDC express CD14 but not CD1a. The CD14-negative subset of DDC were shown to be as potent stimulators of allogeneic mixed lymphocyte reactions as Langerhans cells or blood-derived dendritic cells. However, DDC subsets differed in their ability to support autologous T cell proliferation in response to the mitogenic lectin PHA or bacterial-derived superantigen. In these assays, subsets 1 and 2 were significantly more potent as antigen-presenting cells compared with subset 3. Thus, normal skin contains at least three separate populations of DDC, which have distinctive phenotypic markers and immunologic capabilities.

Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.

The relative contribution of dermal-derived immunocompetent cells to the overall immunologic response in skin has been hampered by the lack of appropriate isolation techniques. In this report, we provide a purification schema that reliably yields highly purified populations of dermal dendritic cells (DDC). These cells are motile, express high levels of class II MHC antigens that decorate their cytoplasmic dendritic processes, and lack numerous B cell, T cell, and natural killer cell antigens. Using a broad panel of 45 different antibodies, an extensive phenotypic analysis was completed, revealing distinctive profiles for subsets of DDC. Despite homogeneous light scatter profile and cytologic appearance, three subsets of DDC could be distinguished by phenotypic and functional criteria. All DDC, but not epidermal Langerhans cells, express factor XIIIa. By triple color cell staining the relative distribution of factor XIIIa positive DDC is as follows: subset 1, 65% to 70% of total DDC express neither CD1a nor CD14; subset 2, 15% to 20% of total DDC express CD1a but not CD14; and subset 3, 10% to 15% of total DDC express CD14 but not CD1a. The CD14-negative subset of DDC were shown to be as potent stimulators of allogeneic mixed lymphocyte reactions as Langerhans cells or blood-derived dendritic cells. However, DDC subsets differed in their ability to support autologous T cell proliferation in response to the mitogenic lectin PHA or bacterial-derived superantigen. In these assays, subsets 1 and 2 were significantly more potent as antigen-presenting cells compared with subset 3. Thus, normal skin contains at least three separate populations of DDC, which have distinctive phenotypic markers and immunologic capabilities.

Heterogeneous populations of class II MHC+ cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogous to Langerhans cells.

Little is known regarding the identification, classification, and function of class II MHC+ dendritic cells in the perivasculature of human connective tissues, such as the dermis. We developed a method for preparing papillary dermal cell suspensions from human keratome strips. Among the class II MHC+ populations of the dermis identified using triple color flow cytometry, cells of monocyte/macrophage lineage (CD45+ CD1- CD11b+ CD11clo-mid CD32+ CD36+ or - CD11a-) and mesenchymal cells of non-bone marrow origin (CD45-) were identified and characterized. Another distinct class II MHC+ subset was identified, which expressed a number of features analogous to epidermal Langerhans cells (LC) and other dendritic APC. These were a numerically minor population comprising only 2.7% +/- 1% (n = 7) of dermal cells. Like LC, they express HLA-DR, CD45, CD1a (albeit at a lower level of expression), CD1c, and CD32 and lack constitutive CD11a or ICAM-1. In contrast to LC, this dermal CD1a+CD1c+ subset expresses CD1b, CD11b, a higher level of CD11c, and intracytoplasmic factor XIIIa. Alloantigen presentation by unfractionated dermal cells was reduced by prior removal of this CD1b+ subset to the same degree achieved by removal of the entire DR+ population (20% of dermal cells), indicating that this was the critical DR+ subset. Cocultures of CD4+ T lymphocytes with cells sorted by flow cytometry into CD1c+DR+, CD1c-DR+ and DR- dermal cell subsets positively identified the CD1c+DR+ population as the most potent of potential APC subsets in human dermis. Thus, in distinction to other dermal macrophage and mesenchymal subsets with elongate morphology, the CD1aloCD1b,c+CD11c(hi)CD11b+CD32+DR+ population in human dermis is highly analogous to cells of LC/dendritic APC lineage in its phenotype and in its exclusive ability to potently present Ag to T lymphocytes. These studies identify and characterize the APC subset most potent in inducing activation of T cells initially entering the perivasculature of human dermis to be of LC/dendritic APC, and not tissue macrophage, lineage.

Thymic microenvironmental abnormalities and thymic selection in NZB.H-2bm12 mice.

We have taken advantage of an extensive panel of mAb directed to thymic epithelial and nonepithelial stromal cells to examine the expression of these Ag from day 16 of gestation through 6 mo of age in NZB(H-2d), NZB.H-2b, NZB.H-2bm12, C57BL/6(H-2b), and C57BL/6.H-2bm12 mice. In addition, by triple color flow cytometry we have examined the expression of cell surface markers defining distinct stages of intrathymic T cell maturation. New Zealand mice demonstrated three abnormalities. MTS 10 normally stains thymic medullary and subcapsular epithelium. However, New Zealand mice demonstrated striking irregular medullary epithelial cell shape whereas their subcapsular epithelium remained normal. Moreover, New Zealand mice, unlike controls, were found to have MTS 10+ epithelial cells within the cortex. Additionally, MTS 39 and MTS 44, which normally stain reticular cortical epithelium, produced a striking different staining pattern in New Zealand mouse thymus, including the presence of large cortical epithelial cellfree regions, so-called "cortical holes." MTS 33 normally stains cortical thymocytes but in New Zealand mice, there was a severe decrease of MTS 33+ cells. There was also an increase of CD3lowCD4+CD8+ cells in NZB mice, which may include many predeletion thymocytes. Finally, there was a significant increase of CD3highCD4+CD8- cells in NZB.H-2bm12 and C57BL/6.H-2bm12 mice compared with NZB.H-2b and C57BL/6(H-2b) mice. We postulate that these microenvironmental alterations in NZB mice contribute to and reflect altered T cell differentiation, thereby predisposing them to autoimmune disease. Moreover, the increased proportion of CD3highCD4+CD8- cells associated with the H-2bm12 mutation may be involved in the remarkably different profiles of disease between NZB.H-2b and NZB.H-2bm12 mice.

A multiparameter flow cytometric method to study surface molecules involved in interactions between subpopulations of cells

The interactions between T and B lymphocytes are mediated by several antigen-independent adhesion molecules including LFA-1/ICAM-1 and CD2/LFA-3. Recently new pairs of adhesion molecules involved in T and B interactions have been described: CD28/B7, CD5/CD72 and CD45RO/CD22. In order to study those heterotypic adhesion events, the phenotypes of the subpopulations as well as new potential adhesion molecules involved in conjugate formation, we have developed a flow cytometric method which analyses conjugate formation between T and B cells. The two types of cells were loaded with two vital intracellular dyes: human T lymphocytes purified from blood or tonsils were labelled with BCECF-AM (green fluorescence) and the B lymphoblastoid cell line, RPMI 8866 was labelled with Indo-1-AM (blue fluorescence). The two labelled cell populations were mixed, gently centrifuged for 5 min and then incubated at 37°C in a waterbath for 5 min. The cells were then gently resuspended by inversion and analysed with a double laser flow cytometer. This method permitted us to discover new molecular interactions since preincubation of the two populations with monoclonal antibodies directed against some surface molecules inhibited conjugate formation. As an example, using this technique we found that the low affinity IgE receptor, CD23 and the CR2/EBV receptor are involved in T cell/B cell adhesion and can therefore be considered as a new pair of adhesion molecules. This method also seems to be applicable to recombinant cells bearing a single adhesion molecule such as LFA-1 and ICAM-1. A particular advantage of the two intracellular dyes we used is that they are compatible with the dyes commonly used for classical simultaneous triple colour immunofluorescence (phycoerythrin and CyChrome). We were thus able to determine the subpopulations involved in forming conjugates and we found that T-B conjugates were preferentially formed by CD4, CD45RO positive T cells, which are believed to be the memory T lymphocytes.

7-Amino-4-methylcoumarin-3-acetic acid-conjugated streptavidin permits simultaneous flow cytometry analysis of either three cell surface antigens or one cell surface antigen as a function of RNA and DNA content

We have investigated the use of the new coumarin dye AMCA (7-amino-4-methylcoumarin-3-acetic acid), as a fluorochrome for multiple color fluorescence analysis by flow cytometry. AMCA is now commercially available as a streptavidin conjugate. Its excitation wavelength is optimal at 351/364 nm with a UV argon ion laser and it emits optimally in the blue at 450 nm. AMCA-conjugated streptavidin yields a fluorescence intensity comparable to that obtained with FITC-streptavidin. The emission spectrum of AMCA does not interfere with that of FITC and phycoerythrin and thus permits triple color analysis of cell surface antigens without requiring the need for important electronic compensation. A unique feature of AMCA conjugates was the possibility to determine the expression of a surface antigen as a function of cellular RNA and DNA levels stained with acridine orange. AMCA conjugates will thus facilitate multiple color analysis by flow cytometry.

Double and triple staining methods for studying the proliferative activity of human B and T lymphoid cells

New methods for double and triple colour labelling using monoclonal antibodies to the proliferation-associated markers 5-bromo-2′-deoxyuridine (BrdU) and Ki67 are described. In order to make incorporated BrdU accessible to most anti-BrdU antibodies, mild denaturation of the DNA is needed, and this is usually obtained by exposing the cells to acid or base. This procedure destroys most cellular antigens, including nuclear TdT and Ki67. In this study we show that fixation in cold methanol, instead of 70% ethanol, for 30 min followed by immersion in 7 × 10 −3 N NaOH for 10–15 s allows BrdU staining with the simultaneous detection of nuclear, cytoplasmic and membrane antigens as well as preservation of morphological detail. This method is optimal for detection of nuclear Ki67 and TdT. These reagents together with antibodies to membrane antigens can be included in triple colour labelling using second layers conjugated to FITC, TRITC and colloidal gold. With these methods it is now possible to characterize the phenotype of dividing cell populations such as precursors in central lymphoid tissues and germinal centre blasts in peripheral lymphoid organs.

A rapid multicolor Western blot

A multicolor Western blotting technique was developed, by which different kinds or different subtypes of interfon were identified with different colors on a single Western blot. This was achieved by sequentially applying different sets of probing antibodies, enzyme-conjugated developing antibodies and enzyme substrates to detect each of the two or more types of interferon on the Western blot. An improved and much faster method of obtaining the same result by the simultaneous application of more than one kind of probing antibodies, the simultaneous application of a mixture of different enzyme-conjugated developing antibodies followed by successive application of different substrates was also described. In addition, a combination of the sequential and simultaneous techniques was used to produce a triple color Western blot.

Radiative unstabilities in some triple electronic colour centre systems in ionic crystals

The principal unstability of some polyvalent triple impurity systems against the possibility of radiative tunnel transitions (RTTs) is discussed. As an example, a three-centre system of neutral, negatively charged and positively charged F-, F′- and α-colour centres in an alkali halide crystal is considered. If the distance between F- and F'-centres is much smaller than the F'-α distance then, along with a very slow RTT process, F′ + α → 2F, a much faster RTT process F′(h) + F(1) → F(h) + F′(1) is also a possible one. Here, h and 1 denote the relatively higher and lower energy position of the trapping level for an extra electron at two F-sites caused by their different distances from the charged α-centre. This type of radiative unstabilities could be relatively significant in areas of defect clustering.

Genetical and Biochemical Studies on the Red and Yellow Eye Pigments of &lt;i&gt;Drosophila melanogaster&lt;/i&gt;

Single and double or triple eye color mutants of Drosophila melanogaster were used for the experiments. The relative amounts of eye pigments, extracted from heads without proboscis by means of“double extraction”method, were estimated by photometrical measurement. Several kinds of pteridines, which were contained mainly in eyes and testes, were separated by paper chromatography and their amounts were measured fluorometrically. Their relative amounts could be estimated by comparing them with each other.The eye pigments of Drosophila are composed of three kinds, red, yellow and brown. Red and yellow pigments have been proved to be pteridine derivatives, and a metabolic pathway from yellow to red was suggested on the basis of experimental results. A metabolic relationship between several pteridines was indicated on the basis of the influence of various mutant genes on the amount of pteridines.Since pteridines are located in eyes and in testes, a remarkable sexual difference was detected; their total amount in male bodies was about 20 times as large as in female bodies. The genic actions of both w and bw strains are assumed to block up the supply of the common pteridine precursor. The route of supply is more or less depressed by the action of ca, ry and Hnr-3 genes. The homozygous Hnr-3 fly was considered to have incomplete conversion of sepiapterin to tetrahydropterin, because of reduced activity of pterine reductase. The cl and Hnr-1 genes inhibit considerably the transformation of tetrahydropterin into red pigment, but the se gene blocks it completely. From all results, a scheme on pteridine metabolism of Drosophila was devised.