A pilot study of combination anti-cytokine and anti-lymphocyte biological therapy in rheumatoid arthritis.

BACKGROUND: Immunological tolerance in humans using anti-T-cell monoclonal antibodies (mAbs) may be hampered by a pro-inflammatory microenvironment. All clinical trials of such therapies in rheumatoid arthritis (RA), however, have selected patients with active disease at baseline. Concurrent neutralization of inflammation with a TNFalpha antagonist should maximize the potential of anti-T-cell mAbs to induce tolerance in RA. AIM: To evaluate the safety of combining a TNFalpha antagonist and CD4 mAb in RA. DESIGN: An iterative pilot study focused on the safety of such combination therapy. METHODS: Eight poor prognosis, seropositive RA patients were treated with combined CD4 and TNFalpha blockade. Prolonged CD4 blockade was achieved with a humanized mAb, and TNFalpha blockade with a p55 TNF receptor fusion protein. RESULTS: There was a low incidence of classical first-dose reactions to the CD4 mAb, possibly reflecting concomitant TNFalpha blockade. An unusual anaphylactoid reaction was seen, however, and one patient developed a probable allergic reaction after several infusions. Skin rashes were common, as previously reported with CD4 mAb monotherapy. No serious infections were documented during follow-up, despite CD4+ lymphopenia in some patients. Most patients appeared to demonstrate improved RA disease control after the study. After 17-49 months after therapy, one patient was in remission, one remained off disease modifying anti-rheumatic drugs and five had stable disease, three on previously ineffective doses of methotrexate. CONCLUSION: We report, for the first time in man, immunotherapy with a combination of an anti-cytokine and an anti-T-cell reagent. We witnessed an unusual first-dose reaction but there were no significant infectious complications.

Leprosy. Recognition and treatment.

Leprosy is a slowly progressive, chronic infectious disease caused by the bacillus Mycobacterium leprae. It is a very serious, multilating and stigmatizing disease in many parts of the world and early diagnosis and therapy is the most important strategy for its control. The skin and peripheral nerves are the most affected organs. It is highly infective, but has low pathogenicity and low virulence with a long incubation period. The geographical distribution of leprosy has varied greatly with time and it is now endemic only in tropical and subtropical regions such as India and Brazil. The diagnosis of leprosy is made from the clinical picture, but must be complimented by skin bacilloscopy and histopathology. Leprosy has a number of distinct clinical presentations. Indeterminate leprosy is frequently the initial form consisting of a few lesions that either evolves into the other forms or resolves spontaneously. Lepromatous leprosy is the more contagious form and affects mainly the skin. In addition, some peripheral nerves may be thickened and other symptoms maybe present. The tuberculid form affects the skin and nerves, although usually there are few lesions. There is also a form borderline between the lepromatous and tuberculoid forms. Current treatment of leprosy involves use of 3 drugs: rifampicin (rifampin); clofazimine; and dapsone. Multidrug therapy aims to effectively eliminate M. leprae in the shortest possible time to prevent resistance from occurring. The duration of therapy was recently reduced from 24 to 12 months. Other treatment options are under evaluation in both preclinical and clinical trials and a number show promise. The combination of rifampicin, ofloxacin and minocycline given as a single dose has been recommended for the treatment of paucibacillar leprosy. Only when physicians, other health workers, and the population in endemic countries become fully aware of, and able to recognize, the disease in its initial phase, will it be possible for therapy to be instituted at the very beginning with either the standard scheme or the newer ones. Intervention at such an early stage will avoid the onset of the more serious signs and symptoms, meaning that leprosy will eventually become a less important public health problem. Therefore, efforts must be made to alert populations at risk and all health workers of the importance of an early diagnosis and treatment in leprosy infection.

Pharmacokinetics of CAMPATH-1H in BMT patients.

BACKGROUND: CAMPATH-1 (CD52)Abs are used in stem-cell transplantation for prevention of GvHD and rejection. The humanized Ab CAMPATH1H has recently replaced the rat Ab CAMPATH-1G. There was a concern whether it might have a longer half-life in vivo and, possibly, cause prolonged immunosuppression post-transplant. METHODS: Serum samples were collected pre- and post-transplant from patients receiving CAMPATH-1H at 10 mg/day according to two protocols: (A) from Day -5 to Day +4 (total dose, 100 mg), (B) from Day -10 to Day -6 (total dose, 50 mg). The Ab concentrations were measured using an immunofluorescence assay. RESULTS: Lymphocytes were substantially depleted by the second day of treatment and were below 0.1 x 10(9)/L by the day of transplant and for at least 1 month post-transplant. By Day 90 there was a greater recovery in Group B, to a median of 0.32 x 10(9)/L compared with 0.25 x 10(9)/L in Group A. By Day 180, both groups had recovered to approx 0.52 x 10(9)/L. Serum concentrations of CAMPATH-1H on the day of transplant were well above the level necessary for opsonization of lymphocytes. The peak Ab concentration was 6.1 micro g/mL in Group A and 2.5 micro g/mL in Group B. CAMPATH-1H could be detected in Group A for 23 days post-transplant, significantly longer than in Group B (11 days). The terminal half-life in the two groups was similar (range 15-21 days) and contrasts with the half-life of < 1 day previously estimated for CAMPATH-1G. There were no cases of graft failure and the incidence of GvHD was similar in the two groups. DISCUSSION: The humanized Ab CAMPATH-1H appears to persist in the circulation for longer than the original rat Ab CAMPATH-1G. This might contribute to delayed lymphocyte recovery and prohibit the use of early donor-lymphocyte infusions. A short course of treatment given early pre-transplant is likely to be preferable to the extended course given both pre- and post-transplant.

Enhancement in the histological diagnosis of leprosy in patients with only sensory loss by demonstration of mycobacterial antigens using anti-BCG polyclonal antibodies.

This study was undertaken to assess whether the immunoperoxidase technique using anti-BCG serum is able to confirm the diagnosis of early leprosy among patients whose unique clinical manifestation is a localized area of sensory loss, in a higher proportion than the routine mycobacterial staining methods, namely hematoxylin-eosin and Wade. The study was held in the north of a hyper-endemic area of leprosy, Manaus, Amazonas (Brazil). Fifty-one paraffin-embedded skin biopsy blocks were retrieved and processed for the immunohistochemical study, by means of anti-BCG polyclonal antibodies for the detection of mycobacterial antigens. The routine stains confirmed the leprosy diagnosis in 17% of the cases, while the immunostaining method confirmed it in 47%. The McNemar test showed that the observed difference between these two techniques was statistically significant (p = < 0.05). In the same way, 50 blocks of skin conditions considered in the differential histopathological diagnosis of early leprosy were processed for the immunohistochemical test to analyze the possibility of false-positive results which occurred in 8 (16%) patients. The study suggests that immunostaining may increase the proportion of the routine histological diagnosis of leprosy in patients who have sensory loss only, even while using biopsies obtained in fieldwork conditions. This is very advantageous in hyper-endemic areas and in areas that are in the post-elimination period of leprosy control where sensory loss may be a sentinel sign of the disease.

Distinct histopathological patterns in single lesion leprosy patients treated with single dose therapy (ROM) in the Brazilian Multicentric Study.

This paper aims to describe the histomorphologic features of skin biopsies of single lesion leprosy patients recruited at outpatient clinics in four Brazilian states in the Northeast (Amazonas and Rondonia), Southeast (Rio de Janeiro) and Center-West (Goiás) between October 1997 and December 1998. Patients clinically diagnosed as single skin lesion paucibacillary (SSL-PB) leprosy had a standard 4-mm punch biopsy taken from the lesion before rifampin, ofloxacin, minocycline (ROM) therapy. The features of the cellular inflammatory infiltrates, the presence of nerve involvement and acid-fast bacilli (AFB) were used to categorize SSL-PB biopsies into different histopathological groups. Two-hundred-seventy-eight (93.0%) out of 299 patients had a skin biopsy available. Seven single lesion patients were diagnosed as BL or LL leprosy types (MB) by the histopathological exams and 12 cases were excluded due to other skin diseases. Therefore, 259 patients had skin lesions with histomorphological features compatible with PB leprosy categorized as follows: 33.6% (N = 87) of the biopsies represented well-circumscribed epithelioid cell granuloma (Group 1); 21.6% (N = 56) less-circumscribed epithelioid cell granuloma (Group 2); 12.0% (N = 31) were described as mononuclear inflammatory infiltrate permeated with epithelioid cells (Group 3), and 29.7% (N = 77) had perivascular/periadnexal mononuclear inflammatory infiltrate (Group 4). Minimal/no morphological alteration in the skin was detected in only 8 (3.1%) SSL-PB patients categorized as Group 5, who were considered to have leprosy by clinical parameters. SSL-PB leprosy patients recruited in a multicentric study presented histomorphology readings comprising the whole PB leprosy spectrum but also a few MB cases. These results indicate heterogeneity among SSL-PB patients, with a predominance of well-circumscribed and less-circumscribed epithelioid cell granulomas (Groups 1 and 2) in the sites studied and the heterogeneity of local cellular immune response.

Preliminary experience of allogeneic stem cell transplantation for lymphoproliferative disorders using BEAM-CAMPATH conditioning: an effective regimen with low procedure-related toxicity.

Autologous transplantation has an established role in the treatment of lymphoproliferative disorders, but allogeneic transplantation remains controversial. In an attempt to reduce the high procedure-related mortality reported with allografting in lymphoma, we have used BEAM (BCNU, etoposide, cytarabine and melphalan), a standard conditioning regimen for autologous transplantation. As BEAM may be insufficiently immunosuppressive to permit durable engraftment in the allogeneic setting, patients received additional pretransplant immunosuppression with the anti-CD52 antibody CAMPATH-1G from day -5 to day -1. Twelve patients (median age 46 years) underwent allogeneic transplantation for lymphoma (n = 11) or chronic lymphocytic leukaemia (n = 1) from HLA-identical (n = 9) or mismatched (n = 3) sibling donors. Cyclosporin A and methotrexate were used as graft-versus-host disease (GVHD) prophylaxis. One patient died of progressive lymphoma at day +12, the remaining 11 patients engrafted rapidly, with eight demonstrating full donor chimerism. One patient had an episode of rejection and received a further stem cell infusion with sustained recovery. Only one patient developed GVHD (grade I). The low incidence of acute GVHD may be in part related to persisting levels of in vivo CAMPATH-IG at the time of transplantation. Of 11 evaluable patients, nine achieved complete remission (CR), and a further patient achieved CR after donor lymphocyte infusion at 5 months. Our preliminary experience is that this regimen was well tolerated with a low risk of GVHD and appears no more toxic than a BEAM autograft. Further follow-up is required to see whether the low incidence of GVHD impacts upon relapse risk.

Single lesion paucibacillary leprosy: baseline profile of the Brazilian Multicenter Cohort Study.

In Brazil, there is little information about the clinical and epidemiological characteristics of paucibacillary, single skin lesion leprosy patients (SSL-PB). Only recently has the official notification system distinguished leprosy patients with a single lesion as a clinical entity, for whom the single-dose ROM (rifampin, ofloxacin and minocycline) regimen has been recommended. In this paper, we describe the baseline clinical features and the immunological background of a multicenter cohort of SSL-PB leprosy cases enrolled between December 1997-1998. Patients were recruited at health centers located in the following regions: Southeast = Rio de Janeiro; North = Amazon and Rondônia states and Center-West = Goiás state. Eligible cases were newly detected, untreated single-lesion leprosy patients without thickened nerve involvement, and were assessed by clinical, bacilloscopic and histopathological exams. The Mitsuda skin test and anti-PGL-I serology (ELISA) were also performed. Of the 299 SSL-PB leprosy patients, 259 (86.6%) fulfilled the criteria for single-dose ROM intervention. Our results showed that patients recruited from different sites had similar features, considering the clinical and immunological profiles. There was a predominance of adults (mean age 32.4; S.D. = 16.0), and a BCG scar was detected in 76.7% of the children (< or = 15 years old). Only 7 cases were diagnosed as the multibacillary type, representing less than 3% of the patients being misclassified. Our data indicate that in Brazil SSL-PB case ascertainment based on clinical and bacilloscopic criteria can be accurately defined under a routine control program; 75.0% of SSL-PB cases were Mitsuda positive (> or = 5 mm) and seropositivity for anti-PGL-I was detected in 17.3% of the patients. These data are compatible with effective cell-mediated immunity and low bacillary load, suggesting favorable clinical outcomes for most SSL-PB participants of this cohort.

Phase I study of an engineered aglycosylated humanized CD3 antibody in renal transplant rejection.

BACKGROUND: The potential therapeutic benefits of CD3 monoclonal antibodies, such as OKT3, have been limited by their immunogenicity and their propensity to activate a severe cytokine release syndrome. This has constrained the clinical use of OKT3 to the treatment of acute rejection episodes of organ allografts. METHODS: We have humanized a rat CD3 antibody and created a single amino acid substitution in position 297 of the IgG1 heavy chain to prevent glycosylation and, consequently, binding of the therapeutic antibody to Fc receptors and to complement. This antibody has been given as first line antirejection therapy in nine kidney transplant recipients with biopsy-proven acute rejection episodes. RESULTS: None of the patients demonstrated any antiglobulin response nor any significant cytokine release syndrome. Seven of the nine showed evidence of resolution of their rejection, although some patients experienced re-rejection. CONCLUSIONS: These findings suggest that CD3 antibodies can be engineered to lose their toxicity while retaining their potency as immunosuppressants. Nonactivating humanized CD3 monoclonal antibodies now merit further investigation in the management of transplant patients and in therapy of autoimmune diseases.

Anti-globulin responses to rat and humanized CAMPATH-1 monoclonal antibody used to treat transplant rejection.

BACKGROUND: Antiglobulin responses are a significant limitation to the repeated use of murine monoclonal antibodies for treatment of transplant rejection. It is hoped that these might be largely overcome by using antibodies genetically engineered to resemble human antibodies. METHODS: We have compared the responses in patients treated with the CD52 monoclonal antibodies CAMPATH-1G (rat IgG2b) or its humanized derivative, CAMPATH-1H (human immunoglobulin G1). RESULTS: A majority of patients (15 of 17) made responses to the rat antibody, but there were no detectable responses to the humanized antibody (0 of 12). CONCLUSIONS: Although anti-idiotype responses are theoretically possible against humanized therapeutic antibodies and are especially likely to be provoked by cell-binding antibodies, these data show that humanization offers a significant reduction in immunogenicity, potentially allowing repeat courses of treatment.

Anti-adhesion molecule therapy as an interventional strategy for autoimmune inflammation.

Functional inactivation of leukocyte adhesion molecules has been used to intervene in the development of tissue injury in experimental models of postperfusion infarction as well as autoimmune inflammation. We investigated the use of humanized monoclonal antibodies (mAb) against CD18 in the treatment of five patients with vasculitic tissue injury sufficient to threaten infarction or gangrene. The treatment was monitored in three ways: (i) whole-body gamma camera scintiscanning of autologous indium-labeled PMN, (ii) an index of the therapeutic inhibition of adhesion derived from comparison pre, during, and post mAb treatment of the ability of patients' PMN to be aggregated after activation by fMLP, and (iii) flow cytometric analysis of PMN CD18 expression. Four of five patients given anti-CD18 at 20 mg/day for up to 3 weeks showed prompt clinical improvement, with healing of the ulceration and restoration of limb function within 4 weeks, which was sustained. The fifth patient, who was not doing well clinically, decided to withdraw from all active treatment: at autopsy there was no evidence of the underlying vasculitis evident pretreatment. Our findings suggest that anti-adhesion molecule treatment might be an effective immediate treatment in severe vasculitis especially when tissue viability is threatened by progressive infarction and/or development of gangrene.

Early diagnosis and treatment of leprosy in intradomiciliary contacts in a high prevalence area: Amazon region.

The importance of dermato-neurological examination of intradomiciliary contacts is well known as an important secondary preventive measure in leprosy control, due to the fact that it allows early diagnosis and treatment. This is an intervention trial in an area of high leprosy prevalence (Manaus/Brazil) where the proportion of contacts examined is low. The aim of the study is to assess whether a simple educational session conducted among patients increases contacts examination and leads to early case detection. The intervention group had examined more contacts (p < 0.05) but, paradoxically, presented fewer new cases than the control group. The authors discuss the probable causes for this unexpected outcome, the advantages of the intervention and other related issues.

Humanized anti-CD4 monoclonal antibody therapy of autoimmune and inflammatory disease.

We have investigated the biological and therapeutic properties of a humanized anti-CD4 MoAb, hIgG1-CD4, in patients with refractory psoriasis and rheumatoid arthritis (RA). hIgG1-CD4 is a modulating, non-depleting MoAb, which induced a first-dose reaction in most patients treated. It provided brief symptomatic relief in both conditions, and psoriasis appeared easier to control with conventional agents after MoAb therapy. At the doses used, hIgG1-CD4 did not synergize therapeutically with the panlymphocyte MoAb CAMPATH-1H (C1H) in patients with RA treated sequentially with both agents. There were no serious adverse effects definitely attributable to therapy. Our results are compared with those of other CD4 MoAb studies, and factors influencing the outcome of therapy are discussed.

Immunosuppression of canine renal allograft recipients by CD4 and CD8 monoclonal antibodies.

A state of tolerance to MHC mismatched allografts can be generated in rodents by treatment with CD4 and CD8 monoclonal antibodies (mAb). In order to transpose this type of therapy to large animals and ultimately to the clinic, a suitable model is required. To this end we have generated a series of mAb to the canine CD4, CD8, and Thy-1 antigens and have tested their ability to prevent rejection of renal allografts. Donor-recipient pairs were selected from a colony of mongrel dogs in which untreated rejection of two haplotype-mismatched kidneys occurred by day 7 (defined as a serum creatinine > 300 mumol/l). Therapy with either the CD4 or the CD8 mAb, using no other immunosuppression, did not prolong graft survival. Depletion of T cells by a Thy-1 mAb prior to surgery only extended graft survival to day 9. However, treating with combinations of mAb up to day 10 (CD4 plus Thy-1; CD4 plus CD8; or CD4 plus CD8 plus Thy-1) prolonged renal allograft function up to 25 days. Combination of the triple mAb therapy with a sub-therapeutic immunosuppressive drug regimen (cyclosporin A plus azathioprine that alone gave a median survival of 15 days) favored survival to a median of 38 days. This protocol also inhibited the antiglobulin response that had curtailed the effects of mAb treatment, opening the way to more extended, and potentially tolerizing, mAb plus drug regimens.

CD4 and CD8 monoclonal antibody therapy in canine renal allografts.

Therapy with CD4 and CD8 monoclonal antibodies was evaluated in dogs which received double-haplotype MHC-mismatched renal allografts. Neither CD4 nor CD8 monoclonal antibodies given alone prolonged allografts survival (creatinine > or = 300 micromol/l) beyond 7 days. However, combined therapy with CD4 and CD8 antibodies given up to day 10 did prolong allograft survival to a median of 14 days. A longer (21 day) course of CD4 and CD8 antibodies did not extend allograft survival further. The effect of prolonged antibody therapy was restricted by the occurrence of both an antiglobulin response and an anaphylactoid reaction to the monoclonal antibody preparation. When the CD4 and CD8 antibodies were combined with a pan-T-cell-depleting Thy-1 antibody, the survival of double-haplotype mismatched allografts was further prolonged (median 16 days). The median survival of single-haplotype mismatched renal allografts on this triple therapy was 21 days, with one surviving to day 36.

CD4 and CD8 monoclonal antibody therapy: strategies to prolong renal allograft survival in the dog.

The value of CD4 and CD8 monoclonal antibody therapy in tolerance induction has been demonstrated in rodent transplant models. In this paper the immunosuppressive potential of CD4 and CD8 monoclonal antibodies for dog renal allografts was evaluated as a preliminary to tolerogenic studies in this large animal model. Monoclonal antibodies were given for a maximum of 10 days after transplantation. Therapy was stopped prematurely following adverse reactions associated with the recipient developing an antibody response against the foreign (rat) therapeutic monoclonal antibody. Blood trough levels of CD4 and CD8 antibodies indicated that saturating doses were achieved. Although neither CD4 nor CD8 alone prolonged allograft survival (rejection by day 7), combination of CD4 and CD8 antibodies resulted in good graft function for a median of 14 days. The effect of removing circulating T lymphocytes was also assessed using a lytic Thy-1 monoclonal antibody. Alone Thy-1 had little effect but, when combined with CD4, the median allograft survival time was increased to 15.5 days. Reduction of the number of circulating T lymphocytes appears complementary to blockade of CD4 for immunosuppression, while blockade of CD4 combined with removal of CD8 also favours allograft survival.