Immune reconstitution following high-dose chemotherapy with stem cell rescue in patients with advanced breast cancer.

The present study examines the nature of humoral and cellular immune reconstitution in 28 patients with advanced breast cancer following high-dose chemotherapy with stem cell rescue. Patients underwent testing of T, B, NK and dendritic cell function at serial time points until 1 year post transplant or until the time of disease progression. Abnormalities in T cell phenotype and function were observed following high-dose chemotherapy that persisted for at least 6-12 months. The vast majority of patients experienced an inversion of the CD4/CD8 ratio and demonstrated an anergic response to candida antigen. Mean T cell proliferation in response to PHA and to co-culture with allogeneic monocytes was significantly compromised. In contrast, mean IgG and IgA levels were normal 6 months post transplant and NK cell yields and function were transiently elevated following high-dose chemotherapy. Dendritic cells generated from peripheral blood progenitors displayed a characteristic phenotype and were potent inducers of allogeneic T cell proliferation in the post-transplant period. The study demonstrates that patients undergoing autologous transplantation for breast cancer experience a prolonged period of T cell dysfunction. In contrast, B, NK, and DC recover more rapidly. These findings carry significant implications for the design of post-transplant immunotherapy.

Effect of melatonin treatment on 24-h variations in responses to mitogens and lymphocyte subset populations in rat submaxillary lymph nodes.

Wistar male rats were injected s.c. with melatonin (30 microg) or vehicle, 1 h before lights off, for 11 days. Ten days after beginning melatonin treatment, rats received Freund's complete adjuvant or its vehicle s.c., and after 2 days, they were sacrificed at six different time intervals throughout a 24-h cycle. The mitogenic effect of lipopolysaccharide (LPS) and concanavalin A (Con A), the activity of ornithine decarboxylase (ODC) and the relative size of lymphocyte subset populations were measured in submaxillary lymph nodes. In control rats, the mitogenic effects of LPS and Con A and ODC activity peaked during the afternoon. Injection of Freund's adjuvant induced a 10-h shift in the diurnal rhythm of the mitogenic effect of LPS to attain maximal values at night. Melatonin pretreatment blunted the daily variations in the mitogenic activity of Con A or LPS and, when given to Freund's adjuvant-injected rats, augmented mesor and amplitude of diurnal rhythm in ODC activity. Maxima in B cell number occurred at night whereas those of T and B-T cell number occurred during the afternoon. During the early phase of immunization tested, the number of B cells augmented and the amplitude of its diurnal rhythmicity increased both after immunization and following melatonin pretreatment. Maxima of 24-h rhythms in CD4+ and CD4+/CD8+ cell populations occurred during the afternoon while those of CD8+ cells occurred at late night. Melatonin significantly augmented CD4+ cell number and decreased CD8+ cell number; it therefore augmented the CD4+:CD8+ ratio. The results suggest that pretreatment with a pharmacological dose of melatonin exerts immunomodulating effects at an early, preclinical, phase of Freund's adjuvant-induced arthritis in rats.

Role of immune modulation in primary HIV infection.

Hydroxyurea inhibits HIV without attacking the virus directly. By inhibiting a cellular enzyme (ribonucleotide reductase) the drug decreases the intracellular concentration of deoxynucleotide triphosphates, thus favoring the incorporation of other drugs, such as the reverse transcriptase inhibitors, into the nascent viral DNA. A large body of data has shown that hydroxyurea can be successfully used during chronic infection. In this manuscript we review the use of hydroxyurea during primary HIV infection. In several independent studies hydroxyurea has been shown to: 1) inhibit HIV in combination with a reverse transcriptase inhibitor and a protease inhibitor as efficiently as with standard highly active antiretroviral therapies; 2) stimulate the immune system by increasing the percentage of naïve cells and the percentage of cells capable of responding to antigens; 3) "cool down" the immune system hyper-activation. The role of hydroxyurea in inducing control of HIV during structured treatment interruptions remains to be clarified.

Four drug-HAART in primary HIV-1 infection: clinical benefits and virologic parameters.

BACKGROUND: From a theoretical standpoint, primary HIV infection (PHI) represents a great chance to modify the natural history of the disease. In this study we purposed a four drugs regimen with zidovudine, lamivudine, ritonavir and saquinavir to treat aggressively the infection and achieve a complete immune reconstitution. METHODS: This is an Italian multicentric open label study. Adult patients with PHI were eligible for the study if they met at least one clinical criterion and one laboratory criterion of the following. Clinical criteria: Signs and symptoms of acute retroviral syndrome within the past 70 days, exposure to HIV-1 within the last 3 months, a preceding negative antibody test within the past 6 months. Laboratory criteria: Detectable p24 antigen with neutralization in serum; detectable HIV-RNA in plasma; indeterminate Western blot test with negative or low positive value HIV antibody in ELISA test. RESULTS: Since April 1997 to April 1999 40 patients with PHI have been enrolled; 80% of this cohort referred symptoms related to acute antiretroviral syndrome. Treatment has been withdrawn in 17 patients (12 for intolerance, 3 for toxicity and 2 for failure). At baseline the mean CD4+ T cells count and CD4/CD8 ratio were 537 (range 55-1287) and 0.58 (range 0.1-1.03) and the mean plasma HIV-RNA level was 5.9 log copies/ml (range 3-7.15). Plasmatic HIV-1 RNA levels of all patients dropped below 200 copies/ml in 68% of patients at week 12, 81% at week 24, 93% after 12 months and 100% after 18 months. Immunological parameters have been improved and have achieved normal range since 6th month. CONCLUSIONS: A rapid virologic suppression and immunological reconstitution are associated with PHI therapy. However early treatment should be weighted against the potential disadvantages such as immediate adverse events (intolerance and drug toxicity) and long term manifestation (metabolic disorders).

Exogenous glucocorticoids alter parameters of early feline immunodeficiency virus infection.

Feline immunodeficiency virus (FIV), a lentivirus, causes progressive immunosuppression and neurologic dysfunction in cats. Glucocorticoids are common therapeutic agents that are also immunosuppressive, and their use might enhance the pathogenic effects of lentivirus infections. Methylprednisolone acetate, a long-acting glucocorticoid, was administered to cats before FIV inoculation, and the course of early infection was monitored. The humoral immune response to FIV was not affected by corticosteroid treatment, but CD8+ cell-mediated antiviral activity was poor in cultures from FIV-infected cats treated with methylprednisolone. Steroid-treated cats had higher plasma viral RNA levels than untreated cats during acute viremia. In contrast, FIV-associated changes in brain stem auditory-evoked potentials were slow to develop in the methylprednisolone-treated cats. Methylprednisolone treatment of cats with established FIV infections appeared to reverse these neurophysiologic changes. These results emphasize the complexity of host-lentivirus interactions and suggest potential advantages and drawbacks of using glucocorticoids in lentivirus infections.

Residual low-level viral replication could explain discrepancies between viral load and CD4+ cell response in human immunodeficiency virus-infected patients receiving antiretroviral therapy.

We report the evolution of chronic infection with human immunodeficiency virus type 1 (HIV-1) in a patient treated with stavudine plus didanosine, whose CD4+ lymphocyte count progressively decreased, despite a sustained plasma viral load <20 copies/mL. After 12 months of therapy, treatment was switched to zidovudine plus lamivudine plus nelfinavir. CD4+ T cell count decreased from 559 x 10(6)/L at month 0 to 259 x 10(6)/L at month 12. Plasma viral load decreased from 21,665 HIV-1 RNA copies/mL at baseline (month 0) to <20 copies/mL after 1 month of therapy with stavudine plus didanosine, and remained below 20 copies/mL until month 12, but always >5 copies/mL. Viral load in tonsilar tissue at month 12 was 125,000 copies/mg of tissue. After the change to triple-drug therapy, the plasma viral load decreased to 5 copies/mL, the CD4+ T cell count increased to 705 x 10(6)/L, and the viral load in tonsilar tissue decreased to <40 copies/mg of tissue at month 24. A low level of HIV-1 replication could explain the lack of immunologic response in patients with apparent virological response.

Granulocyte chemiluminescence activity, antibody production and percentage of CD4(+) and CD8(+) lymphocytes in peripheral blood of offspring of salbutamol-treated pregnant C3H mice.

Preterm delivery is one of the most important problems in obstetric care. One of commonly used treatment of such high risk cases is salbutamol-beta(2) adrenoceptor agonist. The aim of present study was to determine if such treatment causes any changes in neonatal immune system and therefore should be considered in newborn care. The experiments were performed in 4-5- and 6-7-week-old female and male offspring of salbutamol treated C3H inbred mice. In the present study chemiluminescent activity of peripheral blood granulocytes, percentage of CD4(+) and CD8(+) lymphocytes and antibody production were evaluated. A lower number of peripheral blood granulocytes in 6-7-week-old offspring of salbutamol treated mothers was observed, while in the case of younger mice's lymphocytes count in both groups, the differences were not significant as compared to control group. In 4-5-week-old mice a lower percentage of CD4(+), CD3(+) and CD8(+) was evaluated, while in older offspring the percentage of CD4(+) and CD3(+) was higher in the case of the progeny of salbutamol treated mothers. As far as chemiluminescent activity was concerned no differences were found in any of experimental groups. We showed higher IgM production both in male and female offspring of the experimental group and no changes in IgG levels in mice sera. Alterations observed in progeny of salbutamol treated mice might have influence on their further immune system development and function.

Toxicity, biological activity, and pharmacokinetics of TXU (anti-CD7)-pokeweed antiviral protein in chimpanzees and adult patients infected with human immunodeficiency virus.

The purpose of the present study was to evaluate the toxicity and pharmacokinetics of TXU (anti-CD7)-pokeweed antiviral protein (PAP) in human immunodeficiency virus (HIV)-infected chimpanzees and adult patients. At a total dose of 100 microg/kg, TXU-PAP did not cause severe (grade >/= 3) toxicity in any of the four HIV type 1 (HIV-1)-infected or two healthy chimpanzees. The only side effects were a transient elevation of the liver enzyme alanine aminotransferase between days 2 and 14 without a concomitant rise in total bilirubin levels and a decrease in the serum albumin levels between days 1 and 5 without any concomitant weight gain or peripheral edema. TXU-PAP showed favorable pharmacokinetics in chimpanzees with a plasma elimination half-life of 5.1 to 12.0 h and a systemic clearance of 5.8 to 15.1 ml/h/kg. At 2 months after initiation of the TXU-PAP infusions, the HIV-1 burden was reduced to below-detection levels in three of the four chimpanzees, and in the remaining chimpanzee, the HIV burden was <500 RNA copies/ml at 2 weeks but returned to the pretreatment levels by 2 months. TXU-PAP was well tolerated by HIV-1-infected adult patients who received a single 5 microg/kg i.v. infusion of TXU-PAP. TXU-PAP showed very favorable pharmacokinetics in these patients with a relatively long plasma elimination half-life of 12.4 +/- 1.4 h, a mean residence time of 17.9 +/- 2.0 h, and a slow systemic clearance of 2.7 +/- 0.7 ml/h/kg. Concentrations of TXU-PAP required for effective inhibition of HIV-1 replication in preclinical models were achieved in HIV-1-infected patients at the 5 microg/kg dose level without any adverse reactions, and the mean value for AUC was 3059 +/- 721 ng. h/ml. The 1-h postinfusion plasma samples from TXU-PAP-treated patients showed potent anti-HIV activity in vitro and inhibited the replication of HIV in normal peripheral blood mononuclear cells (PBMCs) even at a 1:100 dilution. Although treatment with TXU-PAP at the 5 microg/kg dose level does not provide sustained therapeutic levels, it was capable of reducing the viral burden in six of six patients evaluated. To our knowledge, this is the first report of a clinical pharmacokinetics study of a PAP immunoconjugate in HIV-infected patients. The favorable long plasma elimination half-life of TXU-PAP in combination with its low toxicity provides the basis for further investigation of TXU-PAP as a potential anti-HIV agent.

Lymphocyte subsets as prognostic markers for cancer patients receiving immunomodulative therapy.

Immunogenic features of some malignancies have aroused interest in immunotherapy of cancer. Immunotherapy seems most effective in patients with a small tumour burden, and the focus of immunotherapy trials has, thus, lately been on adjuvant treatment. To enable further development of immunotherapy we need to know more about the mechanisms involved in host defence, especially when the system is influenced by extrinsic factors, that is, immunomodulative agents. T lymphocytes play an important role in the host defence against tumour cells trying to escape from immune surveillance. The mechanisms that regulate the host defence systems are complex, and the influence of extrinsic factors such as immunotherapeutic agents is poorly understood. Most data on lymphocyte subsets in malignant disease originate from melanoma or renal cell carcinoma (RCC) studies, although there are scattered data on lymphocyte subsets also in other malignancies. There are several studies implying that the relative amount of CD4+, CD8+, and natural killer (NK) cells may be important and that, by reducing the tumour burden or by using different therapeutic agents, we can stimulate the host defence. However, only some of these studies imply that these changes can have an impact on clinical outcome and prognosis. The findings of the studies reviewed in this paper are mostly encouraging, but whether the lymphocyte subsets have any value as prognostic markers in patients with malignancies receiving immunotherapy is still unclear. Large randomized immunotherapy trials including an observation arm give an ideal opportunity to recognize those immunological changes that are due to therapy, related to the natural host defence, or whether they have any prognostic value.

The absence of correlation between immunoregulatory T cells and induced lymphoproliferative response in treated B-chronic lymphocytic leukemia patients.

BACKGROUND: Many data suggest T cell functional impairment in B-cell chronic lymphocytic leukemia (B-CLL). The mechanism responsible for this phenomenon is still unresolved. METHODS: In 88 B-CLL patients (RAI II-IV) the relationship between immunoregulatory T cells and PHA induced lymphoproliferative response (LPR) was analysed before and after the therapy. The number of peripheral blood CD3+, CD4+ and CD8+ T lymphocytes was determined by indirect immunofluorescence assay using monoclonal antibodies. LPR was estimated in whole blood culture method. RESULTS: The absolute number of CD3+, CD4+ and CD8+ cells in untreated CLL patients was much higher than in healthy controls (n = 26), but the percentages of these subpopulations, CD4/CD8 ratio and LPR to PHA were significantly (p < 0.00001) decreased. The chemotherapy induced a significant rise of CD3+ and CD4+ percentages (p < 0.006 < p < 0.022 respectively) in comparison to baseline levels, but their levels remained significantly (p < 0.00001) lower than the controls. The CD4/CD8 ratio was also elevated after the therapy (p < 0.048) but remained below the normal value as well. The absolute number of CD3+ and CD4+ T cells were normalized after treatment, while the CD8+ cells were still higher (p < 0.044) than controls. The increase of LPR has been registered after treatment, but it failed to reach the control values. We could not find any correlation between the number of immunoregulatory T cells and induced LPR (r = 0.07, for CD4+; r = 0.09 for CD8+ cells). CONCLUSIONS: These data indicate some profound lymphoid cell defect in CLL patients affecting CD8+ proliferation as well as LPR.

Acute toxicity of an anti-Fas antibody in mice.

By histopathologic examination of various organs in 3 normal strains, C3H/HeN, ICR, and DBA/1J, of mice treated intravenously once with anti-Fas antibody (Jo2), we failed to determine any target organ, except the liver, responsible for the acute lethality induced by the Fas/anti-Fas antibody interaction. However, we could show the presence of Fas-mediated apoptosis in other organs aside from the liver and normal mouse strain differences in susceptibility to anti-Fas antibody. Among these strains, C3H/HeN was the most susceptible to the antibody, followed by ICR and DBA/1J. We observed Fas-mediated apoptosis in the liver, spleen, thymus, lymph nodes, Peyer's patch, intestine, skin, coagulation glands, ovary, uterus, and vagina in all 3 strains and additionally in the epididymides and seminal vesicles in the DBA/1J strain. We also demonstrated that Fas-mediated apoptosis of small lymphocytes in the mantle zone of splenic lymphatic follicles preceded that of the hepatocytes or thymic cells. Since cellular damage was most severe in the liver among all the apoptotic organs in the 3 mouse strains, liver injury induced by anti-Fas antibody is speculated to play a significant role in the death.

The preoperative administration of lentinan ameliorated the impairment of natural killer activity after cardiopulmonary bypass.

The aim of this study was to determine whether the preoperative administration of lentinan, which is used clinically to activate T cell function in cancer patients, prevents the impairment of lymphocyte function after cardiopulmonary bypass (CPB). A total of 25 adults undergoing coronary artery bypass grafting were enrolled in this study. Lentinan (2 mg) was given to 10 randomly selected patients 7 d before surgery, while the other 15 patients were considered as a control. The white blood cell count, percentage of lymphocytes, subsets of lymphocytes, and natural killer cell activity were measured preoperatively, immediately after CPB and 1, 3, and 6 d after surgery. The white blood cell counts and the percentage of lymphocytes were not significantly different between the two groups; however, the percentage of CD4-positive cells in the lentinan group recovered to normal more rapidly than in the control group. Although natural killer cell activity was impaired in the control group after CPB, it maintained a nearly normal level in the lentinan group. The preoperative administration of lentinan for patients undergoing CPB ameliorated the impairment of natural killer activity and promoted the rapid recovery of CD4-positive cells.

Successful treatment of alopecia areata-like hair loss with the contact sensitizer squaric acid dibutylester (SADBE) in C3H/HeJ mice.

A type of hair loss closely resembling human alopecia areata has been described in C3H/HeJ mice. In order to test the assumed analogy with human alopecia areata, we investigated the efficacy of treatment with the contact allergen squaric acid dibutylester. In 12 C3H/HeJ mice with alopecia areata an allergic contact dermatitis was induced and elicited weekly on one side of the back by topical applications of squaric acid dibutylester. Overt hair regrowth was observed only on the treated side of the back in nine of 12 mice. Histopathologic examination revealed a change in the distribution of the inflammatory infiltrate from a dense perifollicular lymphocytic infiltrate around the mid and lower regions of hair follicles in untreated skin to a uniform presence in the upper dermis in treated skin. Immunohistomorphometric studies revealed that treatment with squaric acid dibutylester increased the CD4+/CD8+ ratio from approximately 1:2 in untreated alopecia areata to 1:1 in treated alopecia areata. Additional immunohistochemical investigations showed an aberrant expression of major histocompatibility complex class I, major histocompatibility complex class II and intercellular adhesion molecule 1 on keratinocytes of the mid and lower parts of hair follicles in untreated alopecia areata. In successfully treated skin ectopic major histocompatibility complex class I and II expression was clearly reduced, whereas intercellular adhesion molecule 1 expression showed only minor changes. In conclusion, alopecia areata-like hair loss in C3H/HeJ mice responded to treatment with the contact sensitizer squaric acid dibutylester analogous to human alopecia areata. Moreover, successful treatment changes the aberrant expression of major histocompatibility complex class I and II in a way similar to that observed in human alopecia areata. These observations support the concept that alopecia areata-like hair loss in C3H/HeJ mice can be utilized as an appropriate model for the study of human alopecia areata.

T-cell subsets in schizophrenia: a comparison between drug-naive first episode patients and chronic schizophrenic patients.

T-cell subsets (CD3+, CD4+, CD8+, NK-cells) and the CD4+/CD8+ ratio were measured in 56 schizophrenic patients admitted to hospital with an acute psychosis. Thirty-five patients with chronic schizophrenia and 21 drug-naive first episode schizophrenic patients were compared with 16 healthy controls. T-cell subsets were quantified in the acute state of the illness (day 0), after 7 days of treatment and at the time of discharge. In the acute state, schizophrenic patients showed higher CD3+ and CD4+ cells (p = 0.05) and a higher CD4/CD8 ratio (p = 0.02) than healthy controls, while NK-cells were lower (p = 0.05). In first episode patients, all T-cell alterations normalized during treatment. In the chronic group the ratio remained high, whereas the initially low number of NK-cells normalized over time. These findings, supporting immune system dysregulation in schizophrenia, are discussed in relation to psychopathology, the stage of illness and effects of medication.

Role of macrophage protection in the development of murine AIDS.

Macrophages play a key role in AIDS pathogenesis and thus controlling infectivity and viral replication in these cells is a key issue in any antiretroviral therapy. In the present study, using a murine model of AIDS, we evaluated new therapeutic approaches specifically designed for the protection of macrophages. Based on previous observations, we took advantage of the unique ability of autologous erythrocytes to deliver drugs selectively to macrophages. The antiviral drugs selected were a new homodimer of AZT (AZTp2AZT) and reduced glutathione (GSH). The addition of an oral drug for the protection of lymphocytes (i.e., AZT) was also investigated. C57BL/6 mice infected with the retroviral complex LP-BM5 were treated with GSH-loaded erythrocytes, GSH-loaded erythrocytes plus oral AZT, or GSH/AZTp2AZT-loaded erythrocytes plus oral AZT. The treatments including AZT and erythrocytes loaded with GSH alone or with GSH plus AZTp2AZT provided similar results and were most effective in inhibiting the progression of MAIDS; they reduced splenomegaly, lymphadenopathy, and hypergammaglobulinemia by about 70%, 90% and 83%, respectively, when compared with infected animals at 10 weeks postinfection. Evaluation of BM5d proviral DNA content in infected organs revealed that both treatments were able to almost completely protect most infected animals. They were also able to normalize the blood lymphocyte phenotype and to restore the responses of T and B cells to mitogens significantly. Treatment with GSH-loaded erythrocytes alone did not provide significant results for most parameters investigated, but a marked reduction in proviral DNA content was obtained in infected organs, including the brain. The results reported in this paper confirm the important role of macrophages in retroviral infection and moreover prove that erythrocytes, by selectively protecting these cells, strongly affect MAIDS progression. Furthermore, the combination of GSH- or GSH/AZTp2AZT-loaded erythrocytes with an oral nucleoside analogue (AZT) for the protection of lymphocytes provides additive responses in all the parameters investigated.

Vanadium stimulates immunological responses of chicks.

In a continuation of studies on the interaction of dietary phosphorus (P) and vanadium (V) levels, studies have directed toward an examination of this interaction on the immune system of chicks. Antibody titers to sheep red blood cells (SRBC) were increased at 7 days post-inoculation (PI) by as little as 10 mg V/kg diet in the P-deficient group, while 50 mg V/kg was required in the P-supplemented group. At 14 days PI, only the 50 mg V/kg was significantly higher in both P-deficient and P-supplemented groups. At 21 days PI, vanadium had no significant effect. P-deficiency resulted in a decrease in the percentage of phagocytic macrophages obtained from the abdominal cavity and a decrease in the number of intracytoplasmic SRBC per phagocytic macrophage. These two criteria were increased by vanadium in both the P-deficient and P-supplemented animals. In P-supplemented animals, the CD4/CD8 ratios of lymphocytes obtained from the blood and spleen were increased by the inclusion of 50 mg V/kg diet. The IL-1-like activity of macrophage supernatants was not significantly affected by dietary V, but IL-6 activity was increased. Densitometric analysis of lysates of macrophages isolated from control and V-fed chicks for anti-protein-tyrosinephosphate (PTP) bands indicate that dietary V increased PTP. While the evidence is not clear that there is a P x V interaction in the immune system studies, it is clear that dietary V at the levels used results in a positive immune response of chicks, possibly mediated through increased PTP.

Evaluation of immunomodulatory effects of nisin-containing diets on mice.

The effect of nisin on the immune response of mice was studied. Nisin (in the form of the commercial preparation Nisaplin) was incorporated in the diet of experimental mice which were fed for 30, 75 or 100 days. Short-term administration of diets containing Nisaplin induced an increase of both CD4 and CD8 T-lymphocyte cell counts and also a decrease of B-lymphocyte counts. After prolonged diet administration, T-cell counts returned to control levels. Normal levels of B-lymphocytes were also reached after prolonged administration of the lower (but not the higher) Nisaplin concentration. The macrophage/monocyte fraction isolated from peripheral blood became significantly increased after long-term administration (100 days) of Nisaplin-containing diets in a concentration-dependent way. Although the number of peritoneal cells was not affected by the diets, the phagocytic activity of peritoneal cells decreased after prolonged administration of low (but not high) Nisaplin doses.

Orally administered dextran sulfate is absorbed in HIV-positive individuals.

Preliminary in vivo studies suggested that oral dextran sulfate was poorly absorbed, but investigations were limited by inadequate methods for measuring the drug in the body. To determine absorption in HIV-positive subjects, hydrogenated dextran sulfate, average molecular weight 8000 (Usherdex 8), was orally administered in a short-term (single dose, 4 g/day for 5 days, 7 subjects) and in a long-term study (1 g, 4 times per day for 29 to 335 days, 8 subjects), which was a continuation of the short-term study with the inclusion of an additional subject. When an agarose gel electrophoresis technique with toluidine blue staining was used, the drug was recovered from plasma (67%, peak 2.2 microg/mL) and circulating peripheral blood lymphocyte (PBL) samples (50%, peak 333 microg/L blood) obtained at 5 and 15 minutes and 1, 3, 6, and 24 hours after the first day's dose and from plasma (56%) and PBL samples (38%) obtained 5 minutes after administration on 4 subsequent days in the short-term study. In the long-term study, the drug was found in plasma (67%, peak 2.4 microg/mL) and PBL samples (25%, peak 126 microg/L blood) obtained at monthly visits within 4 hours of the last dose. The drug was found in all urine samples from all subjects in both studies (short-term study, 24-hour samples up to 4 days after the final dose; long-term study, monthly samples within 4 hours of the last dose). In the long-term study, bone marrow preparations from 3 subjects showed metachromatic inclusions present in reticular cells when the cells were stained with toluidine blue, indicating the presence of sulfated polyanions. A significant rise in activated partial thromboplastin time and a drop in platelet count (P < .025) were demonstrated, with thrombocytopenia developing in 3 patients. Mild-to-moderate gastrointestinal disturbances were experienced by 6 subjects in the short-term study and by all subjects in the long-term study. One subject experienced mild central nervous system symptoms in the short-term study. These results indicate that dextran sulfate is absorbed after oral administration; therefore, further studies on its efficacy, particularly in the early stages of the disease, along with additional observations on its toxicity, are warranted.