Geographic heterogeneity of 4 common worldwide cystic fibrosis non-DF508 mutations in Brazil.

Cystic fibrosis (CF) is an autosomal recessive disease caused by at least 750 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of the most common mutation (DF508) in Brazilian patients of European origin is 47%. To determine the frequency of 4 other common CF mutations (G542X, G551D, R553X, and N1303K) in Brazilian patients of European origin, we used direct polymerase chain reaction (PCR) amplification of DNA obtained from dried blood spots on Guthrie cards. The DNA came from 247 non-DF508 chromosomes from 172 Brazilian CF patients ascertained from 5 different states of Brazil. The results show that the 4 mutations account for 17% of the non-DF508 alleles and only 9% of the total number of Brazilian CF alleles. Overall, the frequency of each mutation is different from northern European and North American populations but similar to southern European populations, mainly the Italian and Spanish populations. When Brazilian patients of European origin are grouped according to state of birth, the frequencies of the mutations are significantly different between southern and southeastern states of Brazil. Therefore there are serious implication for risk assessment of DNA-based tests in heterogeneous populations such as Brazilians. Further studies are needed to identify the remaining 44% of CF mutations for the different populations and regions of Brazil.

Thalidomide protects mice against LPS-induced shock

Thalidomide has been shown to selectively inhibit TNF-alpha production in vitro by lipopolysaccharide (LPS)-stimulated monocytes. TNF-alpha has been shown to play a pivotal role in the pathophysiology of endototoxic shock. Using a mouse model of LPS-induced shock, we investigated the effects of thalidomide on the introduction of TNF-alpha and other cytokines and on animal survival. After injecton of 100-350 mug LPS into mice, cytokines including TNF-alpha, IL-6, IL-10, IL-1beta, GM-CSF and IFN-gamma were measured in the serum. Administration of 200 mg/kg thalidomide to mice before LPS challenge modified the profile of LPS-induced cytokine secretion. Serum TNF-alpha levels were reduced by 93 percent, in a dose-dependent manner, and TNF-alpha mRNA expression in the spleens of mice was reduced by 70 percent. Serum IL-6 levels were also inhibited by 50 percent. Thalidomide induced a two-fold increase in serum IL-10 levels. Thalidomide treatment did not interfere with the production of GM-CSF, IL-1beta or IFN-gamma. The LD50 of LPS in this model was increased by thalidomide pre-treatment from 150 mug to 300 mug in 72 h. Thus, at otherwise lethal doses of LPS, thalidomide treatment was found to protect animals from death.

Nucleotide sequence of the P1 region of foot-and-mouth disease virus strain O1 Caseros.

It has been shown that variation of antigenic site I in VP1 of foot-and-mouth disease virus (FMDV) plays an important role in the antigenic diversification of this virus. However, the O1 Campos strain is able to efficiently cross-protect cattle against the O1 Caseros strain, despite having a different sequence in the site I. In this paper we report and compare the P1 coding region for the capsid proteins of FMDV O1 Caseros and O1 Campos. The deduced amino acid sequence showed a total of 31 amino acid differences. Eight of them are located in surface-exposed loops that have been implicated in antigenic sites. This study should help to identify additional sites to be considered in the development of a new generation of FMDV vaccines.

Foot-and-mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3AB1 nonstructural protein.

Foot-and-mouth disease (FMD) vaccines induce antibodies against structural and some nonstructural proteins present in vaccine preparations. To differentiate between FMDV-infected and vaccinated animals, we developed immunochemical assays capable of detecting antibodies against a FMDV nonstructural protein. Recombinant nonstructural 3AB1 protein was expressed in E.coli and in insect cells and used to detect anti-3AB1 antibodies. ELISA and Western blot analysis showed that sera from cattle infected with FMDV reacted with recombinant 3AB1 protein whereas sera from cattle which had been vaccinated against FMDV, mock-infected, or infected with different bovine viruses did not recognize the 3AB1 protein. In contrast, anti-virus infection associated antigen (VIAA) antibodies were present in both FMDV-infected and vaccinated animals. Detection of anti-3AB1 antibodies in sera of experimentally infected cattle obtained between 7 and 560 days postinfection indicated that immunological tests based on the detection of recombinant 3AB1 protein could be used for the diagnosis of FMDV infection.

Thalidomide protects mice against LPS-induced shock.

Thalidomide has been shown to selectively inhibit TNF-alpha production in vitro by lipopolysaccharide (LPS)-stimulated monocytes. TNF-alpha has been shown to play a pivotal role in the pathophysiology of endotoxic shock. Using a mouse model of LPS-induced shock, we investigated the effects of thalidomide on the production of TNF-alpha and other cytokines and on animal survival. After injection of 100-350 micrograms LPS into mice, cytokines including TNF-alpha, IL-6, IL-10, IL-1 beta, GM-CSF and IFN-gamma were measured in the serum. Administration of 200 mg/kg thalidomide to mice before LPS challenge modified the profile of LPS-induced cytokine secretion. Serum TNF-alpha levels were reduced by 93%, in a dose-dependent manner, and TNF-alpha mRNA expression in the spleens of mice was reduced by 70%. Serum IL-6 levels were also inhibited by 50%. Thalidomide induced a two-fold increase in serum IL-10 levels. Thalidomide treatment did not interfere with the production of GM-CSF, IL-1 beta, or IFN-gamma. The LD50 of LPS in this model was increased by thalidomide pre-treatment from 150 micrograms to 300 micrograms in 72 h. Thus, at otherwise lethal doses of LPS, thalidomide treatment was found to protect animals from death.

Effect of rhuIFN-gamma treatment in multibacillary leprosy patients.

Previous studies have shown that when multibacillary leprosy patients were treated with recombinant human interferon gamma (rhuIFN-gamma) for 6-10 months there was an accelerated reduction in the number of acid-fast bacilli in the skin at the site of injection as well as an accelerated bacillary reduction at distal sites. However, this favorable out-come of IFN-gamma treatment was associated with the development of erythema nodosum leprosum (ENL). The present study was undertaken to investigate whether rhuIFN-gamma-induced bacillary clearance could be disassociated from the induction of ENL. rhuIFN-gamma was administered together with thalidomide and conventional multidrug chemotherapy to newly diagnosed leprosy patients. During treatment with this combination of drugs, the mean reduction in bacterial load was the same as the reduction observed with chemotherapy alone. Moreover, the inclusion of thalidomide in the treatment regimen was associated with a low frequency of ENL episodes. A second group of leprosy patients, who had already completed 2 years of chemotherapy, were treated with rhuIFN-gamma only. In those patients who were skin bacilli negative, ENL did not occur during rhuIFN-gamma treatment. In contrast, in bacilli-positive patients the frequency of ENL during rhuIFN-gamma treatment was higher, as was the occurrence of local erythema and induration. However, rhuIFN-gamma treatment without concomitant chemotherapy did not result in a reduction in the bacterial load in the skin of bacilli-positive patients. These findings, taken together, indicate that rhuIFN-gamma does not, by itself, accelerate bacterial clearance, but requires concomitant chemotherapy to achieve the accelerated reduction in bacillary load. Thalidomide reduces the frequency of IFN-gamma-induced ENL, but also eliminates the IFN-gamma-induced bacillary clearance.

Determination of cystic fibrosis carrier frequency for Zuni native Americans of New Mexico.

The Zuni native Americans of the Southwest have an incidence of cystic fibrosis of approximately one in 333 or seven and one-half times that found for Caucasians. Earlier studies indicated that dF508 was not among the cystic fibrosis mutations causing this disease. Through a collaborative study the R1162X mutation was found on 12 out of 12 cystic fibrosis chromosomes from six Zuni patients. Because of the relative high incidence of cystic fibrosis, we undertook a study to determine the carrier frequency of the R1162X mutation among randomly sampled individuals. We found the carrier frequency in the general population for the R1162X to be 6.7%, a very significant number when compared with the carrier frequency for all cystic fibrosis mutations in the Caucasian population of approximately 4%.

Cellular immune response to Mycobacterium leprae infection in human immunodeficiency virus-infected individuals.

The immune responses to Mycobacterium leprae and other mycobacterial antigens were studied in 11 leprosy patients with concurrent human immunodeficiency virus type 1 (HIV-1) infection. Three patients manifested borderline lepromatous leprosy, and eight patients had borderline tuberculoid (BT) leprosy. Despite the low CD4+ T-cell count in the peripheral blood, no histologic or phenotypic change in the cellular infiltrate in either the lepromatous or tuberculoid lesions was observed when compared with HIV-1-negative patients. Lepromatous lesions contained heavily parasitized macrophages and few CD8+ T cells. Lesions from the patients with BT leprosy showed extensive CD4+ T-cell infiltration despite a significant reduction in CD4+ T-cell counts in the peripheral blood. No acid-fast bacilli were detected in the tuberculoid lesions. HIV-1 infection did not alter the lack of response in lepromatous leprosy to M. leprae antigens either in vitro or in vivo. In contrast, the skin test response to M. leprae antigens as well as the in vitro lymphoproliferative responses to mycobacterial antigens that are usually seen in patients with tuberculoid leprosy were abrogated in the BT HIV-1+ patients. However, production of gamma interferon in response to the same stimuli was preserved in most of the patients. Analysis of cytokine gene expression showed activation of additional cytokine genes in the unstimulated peripheral blood cells of patients with both leprosy and HIV-1 infections as compared with cells from patients with leprosy alone. These results suggest that granuloma formation in leprosy can be independent of the impaired CD4+ T-cell response of the HIV-1 infection. Furthermore, in HIV-1+ individuals with M. leprae infection, activation of cytokine genes is observed even when the circulating CD4+ T-cell count is significantly reduced.

The influence of thalidomide on the clinical and immunologic manifestation of erythema nodosum leprosum.

Immunologic and clinical manifestations of erythema nodosum leprosum (ENL) and their response to thalidomide therapy were evaluated. Circulating tumor necrosis factor-alpha (TNF alpha) levels were assayed in serum obtained from lepromatous leprosy patients at diagnosis, during multidrug therapy, at the onset of ENL episodes, and during treatment with thalidomide. Patients with systemic ENL demonstrated the highest serum TNF alpha levels, which decreased significantly during thalidomide treatment. Serum TNF alpha in nonreactional patients was associated with mild flu-like symptoms and local inflammatory lesions. Serum interferon-gamma (IFN-gamma) was also elevated in patients with high TNF alpha levels. Thalidomide therapy reduced not only serum TNF alpha levels and the clinical symptoms but also the dermal infiltration of polymorphonuclear leukocytes and T cells. The expression of intercellular adhesion molecule 1 and major histocompatibility complex class II antigens on the epidermal keratinocytes was also down-regulated. These results indicate that the thalidomide-induced alleviation of clinical symptoms of ENL was associated with a reduction of TNF alpha levels.

Effect of cutaneous cell-mediated immune response to rIFN gamma on Mycobacterium leprae viability in the lesions of lepromatous leprosy.

1. Studies were carried out to determine the effect of intra-dermal injections of recombinant human interferon-gamma (rIFN gamma) on the viability of Mycobacterium leprae. Twenty-three untreated and 4 treated multibacillary patients, 12 with lepromatous leprosy (LL) and 15 with borderline lepromatous leprosy (BL), were selected for intradermal administration of rIFN gamma or PPD. Treated patients (LL and BL) had received multi-drug therapy according to the recommendations of the World Health Organization, i.e., rifampicin (600 mg/month), dapsone (100 mg/day) and clofazimine (50 mg/day and 300 mg/month) for 1-4 months. Three daily doses of 10 or 30 micrograms rIFN gamma induced local induration and mononuclear leucocyte accumulation. Bacteria isolated from a punch biopsy of the site 21 days after lymphokine administration were injected into mouse foot pads and evaluated for viability and growth. 2. The local response to rIFN gamma (specific activity 2 x 10(7) units/mg protein) induced a delay or total inhibition of M. leprae growth in the mouse foot pad, indicating that the cellular response to the antigen reduced local M. leprae viability. The extent of reduction in viability depended on the dose of rIFN gamma injected and the extent of local induration induced by the lymphokine. With a vigorous cell-mediated immune response growth was fully inhibited. 3. A similar but less extensive effect on M. leprae viability was observed in response to the local injection of 5 units in 0.1 ml of purified protein derivative of tuberculin (PPD).

Effect of cutaneous cell-mediated immune response to rIFNy on mycobacterium leprae viability in the lesions of lepromatous leprosy / Efeito da célula cutanea - resposta imune intermediária para rIFNy sobre viabilidade de Mycobacterium leprae nas lesöes de lepra lepromatosa

Studies were caried out to determine the effect of intra-dermal injections of recombinant human interferon-gamma (rIFNy) on the viability of Mycobacterium leprae. Twenty-three untreated and 4 treated multibacillary patients, 12 with lepromatous leprosy (LL) and 15 with bordeline lepromatous leprosy (BL), were selected for intradermal administration of rIFNy or PPD. Treated patients (LL and BL) had received multi-drug therapy according to the recommendations of the World Health Organization, i. e., rifampicxin (600 mg/month), dapsone (100 mg/day) and clofazimine (50 mg/day and 300 mg/month) for 1-4 months. Three daily doses of 10 or 30 ug rIFNy induced local induration and mononuclear leucocyte accumulation. Bacteria isolated from a punch biopsy of the site 21 days after lymphokine administration were injected into mouse foot pads and evaluated for viability and growth. The local response to rIFN (specific activity 2 x 10 7 units/mg protein) induced a delay or total inhibition of M. leprae growth in the mouse foot pad, ,indicating that the cellular response to the antigen reduced local M. leprae viability. The extent of reduction in viability depended on the dose of rIFNy injected and the extent of local induration induced by the lymphokine. With a vigorous cell-mediated immune response growth was fully inhibited. A similar but less extensive effect on M. leprae viability was observed in resapo0nse to the local injection of 5 units in 0.1 ml of purified protein derivative of tuberculin (PPD)

Protection conferred by TrpE fusion proteins containing portions of the C-terminal region of capsid protein VP1 of foot-and-mouth disease virus.

Major immunogenic sites of foot-and-mouth disease virus (FMDV) have been mapped to the C-terminal third of capsid protein VP1; we studied the immunogenicity of a series of TrpE-FMDV fusion proteins containing this region of FMDV strain O1 Campos. Fusion protein TrpE-dCN, which contains a dimer of VP1 amino acid sequences consisting of amino acids 200 to 213 linked by a diproline spacer to amino acids 141 to 158 (200-213 approximately P-P-G approximately 141-158), induced the best response. A single inoculation of guinea-pigs with 100 micrograms TrpE-dCN elicited high levels of neutralizing antibodies and protected all the animals against challenge infection with homologous virus. Although the closely related FMDV strains O1 Campos and O1 Caseros induced high levels of cross-protection, TrpE-dCN-vaccinated guinea-pigs were poorly protected against challenge infection with heterologous FMDV strain O1 Caseros. Nucleotide sequence analysis revealed that amino acid differences at residues 149 and 152 were critical for the induction of cross-protection and that neutralizing epitopes not present in TrpE-dCN are likely to be responsible for conferring a high level of cross-protection between FMDV strains O1 Campos and O1 Caseros.

VP1 of serotype C foot-and-mouth disease viruses: long-term conservation of sequences.

The nucleotide sequences of the VP1-coding regions of several isolates of serotype C3 foot-and-mouth disease virus (FMDV) were determined. The deduced amino acid sequences were compared with those of serotype C1 FMDV. The results provide evidence for two different lineages of FMDV C3 and document the potential for both long-term conservation and rapid evolution of FMDV.

[Synthesis of 10S particles in cells infected with aphthovirus]. / Síntesis de partículas 10S en células infectadas con aftovirus.

In the present study, evidence is presented for the existence of a morphogenetic intermediary that may be a precursor of the procapsids in the assembling process. BHK21 clone 13S cells were infected with Aphthovirus A24 (Cruzeiro strain), and pulse-chase experiments were carried out using 3H-leucine. Cytoplasmic extracts were then prepared at appropriate times, and analyzed by sucrose-gradient ultracentrifugation. After preliminary assays (Fig. 1), working conditions were standardized so as to obtain maximal recovery of the morphogenetic intermediary, as well as consistency of results. Only in the presence of DOC-Brij58 and Mg++ could a 10S sedimentation coefficient peak be seen (Fig. 1 a). A heterogeneous zone, with 4,5-5S as sedimentation coefficient, was also observed. The degree of labeling in the region 4,5-5S compared with that in the 10S portion, depends on the time within the infectious cycle when cells were pulse-labeled. Maximal levels for the ratio 10S/4,5-5S are reached when pulse-labeling takes place at the time when the amount of RNA viral synthesis reaches 80% of its total value (Fig. 2). Similar experiments, performed with third passage bovine fetal kidney cells, were confirmatory of the presence of a structure sedimenting at 10S, as well as of a heterogeneous zone of 4,5-5S (Fig. 3). It would appear that assembling of Aphthoviruses is accomplished through an intermediary unit which differs from that found for other Picornaviruses, the latter being the result of the union of 12 pentamers. The capsid of Aphthoviruses, also composed of 60 identical sub-units, would instead derive from the joining of 20 trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Síntesis de partículas 10S en células infectadas con aftovirus. / [Synthesis of 10S particles in cells infected with aphthovirus]

In the present study, evidence is presented for the existence of a morphogenetic intermediary that may be a precursor of the procapsids in the assembling process. BHK21 clone 13S cells were infected with Aphthovirus A24 (Cruzeiro strain), and pulse-chase experiments were carried out using 3H-leucine. Cytoplasmic extracts were then prepared at appropriate times, and analyzed by sucrose-gradient ultracentrifugation. After preliminary assays (Fig. 1), working conditions were standardized so as to obtain maximal recovery of the morphogenetic intermediary, as well as consistency of results. Only in the presence of DOC-Brij58 and Mg++ could a 10S sedimentation coefficient peak be seen (Fig. 1 a). A heterogeneous zone, with 4,5-5S as sedimentation coefficient, was also observed. The degree of labeling in the region 4,5-5S compared with that in the 10S portion, depends on the time within the infectious cycle when cells were pulse-labeled. Maximal levels for the ratio 10S/4,5-5S are reached when pulse-labeling takes place at the time when the amount of RNA viral synthesis reaches 80

of its total value (Fig. 2). Similar experiments, performed with third passage bovine fetal kidney cells, were confirmatory of the presence of a structure sedimenting at 10S, as well as of a heterogeneous zone of 4,5-5S (Fig. 3). It would appear that assembling of Aphthoviruses is accomplished through an intermediary unit which differs from that found for other Picornaviruses, the latter being the result of the union of 12 pentamers. The capsid of Aphthoviruses, also composed of 60 identical sub-units, would instead derive from the joining of 20 trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective gamma interferon production in leprosy

Antigen and mitogen-induced gamma interferon (gamma-IFN) production was studied in peripheral blood mononuclear cells from 34 leprosy patients. 17 of 18 lepromatous leprosy and borderline lepromatous patients (LL and BL) failed to release gamma-IFN in response to specific antigen (Mycobacterium leprae) and displayed reduced responses to mitogen (concanavalin A) stimulation. In contrast, cells from six tuberculoid and borderline tuberculoid patients (TT and BT) produced considerable levels of gamma-IFN under the same experimental conditions. Normal controls failed to respond to M. leprae and most displayed good responses to concanavalin A. Mid-borderline patients (BB) showed intermediate levels of gamma-IFN release. gamma-IFN release by lepromatous patients could be partially restored with purified interleukin 2 and M. leprae antigen but not with interleukin 2 alone.