Active tuberculosis patients have high levels of IgA anti-alpha-crystallin and isocitrate lyase proteins.

SETTING: Mexico City, Mexico. OBJECTIVE: To identify proteins synthetised by Mycobacterium tuberculosis in hypoxic culture, which resemble more closely a granuloma environment than aerobic culture, and to determine if they are recognised by antibodies from patients with active pulmonary tuberculosis (PTB). DESIGN: Soluble extracts from M. tuberculosis H37Rv cultured under aerobic or hypoxic conditions were analysed using two-dimensional polyacrylamide gel electrophoresis, and proteins over-expressed under hypoxia were identified by mass spectrometry. The presence of immunoglobulin (Ig) G, IgA and IgM antibodies against these proteins was determined in the serum of 42 patients with active PTB and 42 healthy controls. RESULTS: We selected three M. tuberculosis H37Rv proteins (alpha-crystallin protein [Acr, Rv2031c], universal stress protein Rv2623 and isocitrate lyase [ICL, RV0467]) that were over-expressed under hypoxia. Titres of anti-Acr and anti-ICL IgA antibodies were higher in patients than in healthy controls, with an area under the receiver operating characteristic curve of 0.71 for anti-ICL IgA antibodies. CONCLUSION: ICL could be used in combination with other M. tuberculosis antigens to improve the sensitivity and specificity of current serological TB diagnostic methods.

Adjuvant treatment with a dialyzable leukocytes extract contributes to maintain HPV-infected women free of low-grade cervical lesions.

PURPOSE OF INVESTIGATION: To investigate if adjuvant treatment with a dialyzable extract of leukocytes (DLE), may help HPV-infected patients with low-grade intraepithelial squamous cervical lesions (LIS) to get free of HPV infection and cervical lesions. MATERIALS AND METHODS: Patients with untreated, low-grade cervical lesions were treated either with surgery (Group A) or with DLE (Group B). Pa- tients with low-grade but recurrent cervical lesions were newly treated with surgery plus DLE (Group C). RESULTS: A decreased or ab- sent cervical lesion correlated with a diminished or absent HPV viral load at one year of treatment (r = 0.6,p <0.05). Seventy-nine percent of Group B but only 50 % of Group C and 38 % of Group A patients were free of cervical lesion after 24 months of treatment (p < 0.05). CONCLUSION: The present data support the benefit of adding DLE as adjuvant for treating HPV-infected women with LIS.

Low-dose amphotericin B and murine dialyzable spleen extracts protect against systemic candida infection in mice.

Candida albicans causes opportunistic systemic infections with high mortality (30%-50%). Despite significant nephrotoxicity, amphotericin (AmB) is still used for the treatment of this serious fungal infection. Therefore, alternative treatments are urgently needed. Dialyzable leukocyte extracts have been used successfully to treat patients with mucocutaneous candidiasis, but their effectiveness in systemic candidiasis has not been evaluated. In this study, low-dose AmB (0.1 mg/kg) plus 10 pg of murine dialyzable spleen extracts (mDSE) were tested in a systemic candidiasis mouse model. Survival, tissue fungal burden, kidney damage, kidney cytokines, and serum levels of IL-6 and hepcidin were evaluated. Our results showed that the combined treatment of low-dose AmB plus mDSE improved survival and reduced kidney fungal burden and histopathology; these effects correlated with increased kidney concentration of IFN- γ and TGF- ß 1, decreased levels of TNF- α , IL-6, and IL-10, as well as high levels of systemic IL-6 and hepcidin. Low-dose AmB and mDSE synergized to clear the infectious agent and reduced tissue damage, confirming the efficacy of a low dose of AmB, which might decrease the risk of drug toxicity. Further studies are necessary to explore these findings and its implications in future therapeutic approaches.

Immunotherapeutic effects of recombinant adenovirus encoding granulocyte-macrophage colony-stimulating factor in experimental pulmonary tuberculosis.

BALB/c mice with pulmonary tuberculosis (TB) develop a T helper cell type 1 that temporarily controls bacterial growth. Bacterial proliferation increases, accompanied by decreasing expression of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS). Activation of dendritic cells (DCs) is delayed. Intratracheal administration of only one dose of recombinant adenoviruses encoding granulocyte-macrophage colony-stimulating factor (AdGM-CSF) 1 day before Mycobacterium tuberculosis (Mtb) infection produced a significant decrease of pulmonary bacterial loads, higher activated DCs and increased expression of TNF-α, IFN-γ and iNOS. When AdGM-CSF was given in female mice B6D2F1 (C57BL/6J X DBA/2J) infected with a low Mtb dose to induce chronic infection similar to latent infection and corticosterone was used to induce reactivation, a very low bacilli burden in lungs was detected, and the same effect was observed in healthy mice co-housed with mice infected with mild and highly virulent bacteria in a model of transmissibility. Thus, GM-CSF is a significant cytokine in the immune protection against Mtb and gene therapy with AdGM-CSF increased protective immunity when administered in a single dose 1 day before Mtb infection in a model of progressive disease, and when used to prevent reactivation of latent infection or transmission.

Enoyl-coenzyme A hydratase and antigen 85B of Mycobacterium habana are specifically recognized by antibodies in sera from leprosy patients.

Leprosy is an infectious disease caused by Mycobacterium leprae, which is a noncultivable bacterium. One of the principal goals of leprosy research is to develop serological tests that will allow identification and early treatment of leprosy patients. M. habana is a cultivable nonpathogenic mycobacterium and candidate vaccine for leprosy, and several antigens that cross-react between M. leprae and M. habana have been discovered. The aim of the present study was to extend the identification of cross-reactive antigens by identifying M. habana proteins that reacted by immunoblotting with antibodies in serum samples from leprosy patients but not with antibodies in sera from tuberculosis (TB) patients or healthy donors (HDs). A 28-kDa antigen that specifically reacted with sera from leprosy patients was identified. To further characterize this antigen, protein spots were aligned in two-dimensional polyacrylamide gels and Western blots. Spots cut out from the gels were then analyzed by mass spectrometry. Two proteins were identified: enoyl-coenzyme A hydratase (lipid metabolism; ML2498) and antigen 85B (Ag85B; mycolyltransferase; ML2028). These proteins represent promising candidates for the design of a reliable tool for the serodiagnosis of lepromatous leprosy, which is the most frequent form in Mexico.

Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes.

It has been shown recently that different genotypes of Mycobacterium tuberculosis induce distinct immune responses in the host, as reflected by variations in cytokine and iNOS expression. Because these molecules are probably regulated by multiple factors in vivo this complex phenomenon was partially analysed by assessing cytokine and iNOS expression by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in an in vitro model of bone marrow-derived macrophages infected with three different M. tuberculosis genotypes: Canetti, H37 Rv and Beijing. Although the three genotypes induced production of iNOS and the different cytokines tested at 24 h post-infection, macrophages infected with the Beijing isolate expressed the highest levels of mRNA for iNOS, interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, IL-12 cytokines and lower levels of IL-10 compared with cells infected with other genotypes. This expression pattern has been associated with infection control, but during infection in vivo with the Beijing genotype it is lost upon progression to chronic phase. The failure to control infection is likely to be influenced by cytokines produced by other cell types and bacterial molecules expressed during the course of disease. Results presented in this work show that each genotype has the ability to induce different levels of cytokine expression that could be related to its pathogenesis during infection.

Interstitial transfer factor as adjuvant immunotherapy for experimental glioma.

Glioblastoma multiform (GBM) is the most common tumour of the central nervous system in humans. Unfortunately its prognosis is poor and because of the lack of efficacious therapies, immunotherapy is a potential treatment. Transfer factors (TF) are low molecular weight dialysable products extracted from immune cells which transmit the ability to express delayed-type hypersensitivity and cell mediated immunity from sensitized donors to nonimmnune recipients. In this study, we determined the efficacy of TF as immunotherapy to treat experimental glioblastoma. We used TF obtained from immunized swine. We evaluated different doses of intratumoral TF (product of 4x10(6), 8x10(5) and 1.6x10(5) cells). The best dose (product of 4x10(6) cells) of TF was also combined with carmustine for experimental therapy in rats with C6 malignant glioma. Modifications in peripheral blood T lymphocyte counts ( CD2+, CD4+, CD8+ and NK) were evaluated by flow cytometry. Cytokine expression in the tumour was assessed by RT-PCR and apoptosis was evaluated using the sub G0 method. Intratumoral TF reduced significantly the tumour size, and increased CD2+, CD4+, CD8+ and NK cell counts, it also increased the percentage of apoptotic tumour cells and the percentage of tumour tissue expressing Th1 cytokines. We observed an additive antitumoral effect when TF was combined with chemotherapy.

[Transfer factor as specific immunomodulator in the treatment of moderate-severe atopic dermatitis]. / Factor de transferencia como inmunomodulador específico en el tratamiento de la dermatitis atópica moderada a severa.

BACKGROUND: Atopic dermatitis is a skin inflammatory disease which has been associated to high levels of IgE, eosinophiles and change of T lymphocytes. The transfer factor is an immunomodulator active substance and decreases the number of inflammatory cells and the severity of the symptoms of atopic dermatitis. OBJECTIVE: To determine the efficacy of the transfer factor as treatment of moderate and severe atopic dermatitis. MATERIAL AND METHODS: Articles related to treatment with transfer factor in the atopic dermatitis were looked up in Medline and EMBASE, and the ones referring to controlled studies in patients with moderate and severe atopic dermatitis in accord to SCORAD. RESULTS: We found seven articles with 121 patients and 88 controls demonstrating significant decrease in the symptoms of the SCORAD index, decreased IgE, and eosinophils in patients treated with transfer factor. CONCLUSIONS: The transfer factor is a choice treatment for moderate and severe atopic dermatitis.

The effect of iron on the expression of cytokines in macrophages infected with Mycobacterium tuberculosis.

Iron is known to play an important role in different bacterial infections and, in particular, in their development. One example is infection with Mycobacterium tuberculosis where iron contributes to growth and survival of the bacteria within the host cell. The majority of studies performed on tuberculosis have focused on the direct effect of iron on bacterial growth; however, little is known about how iron modifies the mycobacterial-host interaction. In order to address this, we have investigated the effect of iron on intracellular growth of M. tuberculosis in J774 macrophages and the molecular mechanisms that are affected during this interaction. We observed that iron modifies intracellular growth of the mycobacteria and that their growth kinetics was modified from that observed for the extracellular situation in the presence of iron. Similarly, when iron was present during the infection, there was a reduced release of tumour necrosis factor-alpha and it was related to a higher number of bacilli inside the host cell and low expression of interleukin-1 (IL-1) and IL-6 mRNA. Hence, this work demonstrates that iron, besides promoting mycobacterial growth, also regulates the relationship between macrophage and bacteria.

Transfer factors as immunotherapy and supplement of chemotherapy in experimental pulmonary tuberculosis.

Problems of logistics, compliance and drug resistance point to an urgent need for immunotherapeutic strategies capable of shortening the current six month antibiotic regimens used to treat tuberculosis. One potential immunotherapeutic agent is transfer factors. Transfer factors (TF) are low molecular weight dialysable products from immune cells which transmit the ability to express delayed-type hypersensitivity (DTH) and cell mediated immunity from sensitized donors to nonimmune recipients. In this study we determined the efficiency of TF as immunotherapy to treat experimental tuberculosis. When BALB/c mice are infected via the trachea with Mycobacterium tuberculosis H37Rv there is an initial phase of partial resistance dominated by Th-1 type cytokines plus tumour necrosis factor-alpha (TNFalpha) and the inducible isoform of nitric oxide synthase (iNOS), followed by a phase of progressive disease characterized by increasing expression of IL-4, diminished expression of TNFalpha and iNOS, and low DTH. Animals in this late progressive phase of the disease (day 60) were treated with different doses of TF (one injection per week) obtained from spleen cells when the peak of immune protection in this animal model is reached (day 21), or with different doses of TF from peripheral leucocytes of PPD + healthy subjects. We show here that the treatment with murine or human TF restored the expression of Th-1 cytokines, TNFalpha and iNOS provoking inhibition of bacterial proliferation and significant increase of DTH and survival. This beneficial effect was dose dependent. Interestingly, murine TF in combination with conventional chemotherapy had a synergistic effect producing significant faster elimination of lung bacteria loads than chemotherapy alone.

Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I.

Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.

Apoptosis in lymph nodes and changes in lymphocyte subpopulations in peripheral blood of pigs infected with porcine rubulavirus.

In a first experiment, five pigs were inoculated intranasally with porcine rubulavirus (PoRV) at 5 days of age and killed 7 days post-infection (pi). In a second experiment, four pigs were infected with the same virus at 17 days of age and killed at 9 or 15 days pi. Control piglets in each experiment received uninfected cell culture supernate. All PoRV-infected pigs developed respiratory and nervous signs, and histological lesions of non-suppurative encephalitis and interstitial pneumonia. All control pigs remained clinically normal and did not have histological lesions. Significantly increased numbers of apoptotic cells were detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) in tonsil and lymph nodes of the pigs infected at 7 days of age and killed at 7 days pi. Significantly increased percentages of CD2(+) and CD8(+) T lymphocytes were also found in peripheral blood of these animals at this time, while the percentages of CD4(+) and MHC class II lymphocytes were significantly reduced. Significantly increased numbers of apoptotic cells were detected in lymphoid tissues of the pigs infected at 17 days of age and killed at 9 days pi. The percentages of CD2(+), CD8(+) and MHC class II lymphocytes in peripheral blood were also significantly increased at this time; the percentage of MHC class II lymphocytes remained elevated at 15 days pi. These results indicate that induction of apoptosis is an important mechanism in the pathogenesis of PoRV infection in young pigs, and that this virus induces changes in lymphocyte subpopulations in peripheral blood.

Immunomodulatory properties of Mycoplasma pulmonis. I. Characterization of the immunomodulatory activity.

For many years, it has been recognized that Mycoplasma infection affects the host's immune system in different ways. In this work, experiments were performed to characterize the influence of Mycoplasma pulmonis infection on various immunological parameters and to follow the kinetics of their variations. A Balb/c mouse model was used to assess hematological evaluations, changes in spleen weight, antibody responses against sheep erythrocytes, neutrophil phagocytosis, colloidal carbon clearance, and anti-Mycoplasma antibody responses. At the hematological level, infected animals were found to have significantly increased total lymphocyte and polymorphonuclear leukocyte counts and an augmentation in spleen weight. Seven days after Mycoplasma infection, antibody responses against sheep erythrocytes were considerably diminished, and at days 7 and 14 after infection, phagocytic activity was also reduced. After 1 week of infection, the colloidal carbon clearance pattern was decreased, and during the whole infectious process, anti-Mycoplasma antibody titers were found to be low. Results from this part of research show a persistent infection that does not resolve in a short period, which is associated with a general dysfunction in the immune system and poor immune responses against several different antigens.

Immunomodulatory properties of Mycoplasma pulmonis. II. Studies on the mechanisms of immunomodulation.

Mycoplasma infection affects the host's immune system in different ways. In this work, a kinetic approach was used to try to determine the mechanisms by which Mycoplasma cause these effects. Experiments were performed using Balb/c mice infected with Mycoplasma pulmonis and several immunological parameters were determined. It was found that at days 10 and 15 post-infection, there were significant changes in the percentages of CD4+ and CD8 + cells, in both peripheral blood and the thymus. Significant sequential increases in concentrations of both IFN-gamma and IL-4 were detected in sera, such that at day 15, there was a peak in IFN-gamma, concentration and at day 38, IL-4 concentration also peaked. By day 46, both IFN-gamma and IL-4 fell to control levels despite continued infection. Delayed hypersensitivity (DTH) was reduced in infected animals compared to non-infected controls. A small recovery in DTH was observed at day 30, which was reduced again by day 40. Altogether, the results show features of a transitional shift from Th1 to Th2 in animals that are ultimately immunologically incompetent (in both cellular and humoral immunity). It appears to be this state of incompetence that allows the microorganism to survive and thus provides an explanation for the chronic state of the disease, which is a characteristic of Mycoplasma infection.

Immunomodulatory properties of Mycoplasma pulmonis. III. Lymphocyte stimulation and cytokine production by Mycoplasma pulmonis products.

Experiments are presented that were performed in order to understand the mechanisms causing these effects on the immune system. Mitogenic effects of Mycoplasma membranes on mouse spleen cells were shown using M. capricolum. The observed mitogenic activity is proportional to the amount of membranes used, as measured by protein content. Separation of T and B cells was performed by two techniques, the anti-Thyl.2 plus complement method and the Dynabead technique. Using the former technique, it was shown that removal of T cells markedly reduced effects of stimulation by mycoplasma membranes, but did not abolish it. The separated cells were still stimulated by PHA, indicating that the preparation still contained T cells. Furthermore, removal of T cells preferentially reduced the PHA response over that of mycoplasma membranes, indicating that mycoplasma membranes stimulate both B and T lymphocytes. The Dynabead system was found to be the more efficient separation technique, and by using it we were able to make the following observations. Inactivated Mp, membranes and culture supernatant stimulated B cells, whereas T cells were only slightly stimulated by inactivated Mp and membranes. There was an increase in proliferation when T cells were incubated with adherent cells from peripheral blood. Finally, we showed that spleen cells from infected animals produce more IL-4 and less IFN-gamma than cells from non-infected animals when stimulated with membranes, inactivated Mp, culture supernatant or phytohemagglutinin. Altogether, these results show that lymphocytes from Mycoplasma-infected animals are directly affected and this effect is probably due to superantigen-like molecules from M. pulmonis.

Connectivity patterns in tuberculosis and leprosy patients are indistinguishable from that of healthy donors.

Connectivity, the self-defined interactions between antigen-recognising molecules in a network system can in part be assessed by measuring the reactivity of a given serum against an ordered set of immunoglobulin (Ig)G F(ab')2 fractions, separated by means of isoelectric focusing so that, the serum reactivity against the whole set of fractions defines a characteristic pattern of connectivity. Deviations from the normal condition (healthy donors) have so far been documented for two autoimmune diseases: systemic lupus erythematosus (SLE) and pemphigus vulgaris, as well as for human immunodeficiency virus (HIV)-1 infection. We tested here if bacterial infections lead to alterations in connectivity. In addition, we wanted to test if two antigenically related bacteria would produce similar or otherwise distinctive connectivity patterns. Connectivity analysis was applied on the sera from tuberculosis and leprosy patients and the sera from healthy donors were used as control. No statistically significant differences between the three groups studied were found. These results have implications for theories that set the origin of autoimmune diseases in microbial infections. To the best of our knowledge, this is the first attempt to analyze the connectivity status in bacterial infections.

[Safety and efficacy of treatment for severe atopic dermatitis with cyclosporin A and transfer factor]. / Seguridad y eficacia en el tratamiento de la dermatitis atópica severa con ciclosporina A y factor de transferencia.

BACKGROUND: The atopic dermatitis is a chronic skin disease that appears in patients with a personal or family history of allergic asthma and rhinitis. It is associated to the specific activation of a gene group. In most instances, the response to the conventional treatment is adequate. The are cases, though, know as refractory, where that is not the case. The study of two therapeutic alternatives, Transfer Factor (TF) and Cyclosporin A (CyA), was elaborated for this type of patients. MATERIAL AND METHODS: Patients with severe refractory AD were studied, being admitted to the Allergic Service to the ISSSTE Lic. Adolfo López Mateos, ISSSTE, between September 1997 and june 1998. They were randomly divided in two groups. The first one was subjected to CyA, on a 4 mg/kg/day dosage, with monthly surveillance of kidney and hepatic functions and blood pressure twice a week. Group two was subjected to TF, as follows: one unit every third day for the first week, two units per week for the next three weeks and one monthly unit to complete six months. Initial and final clinical and immunologic testing was performed on both groups (eosinophils, total IgE, CD4 and CD8). RESULTS: Six patients included group A, and 12 patients in group B. Both groups showed a significant statistic reduction in the total eosinophils count, without an statistic difference between them. None showed changes in the total IgE. CyA reduced the CD4 levels, while the TF increased the levels of CD8 cells, both with a p < 0.05. Both groups showed clinical improvement satistically significant, but no differences with a p > 0.05 appeared between them. Tolerance to the treatments was adequate, and there was not need to suspend the treatment in any case. Only three patients showed hypertricosis and other one presented headaches, with CyA. CONCLUSION: Both treatments showed therapeutic benefits in the treatment of patients with severe refractory AD, with similar immunologic improvement. Both drugs present different action mechanisms, so their joint application could offer clinical benefit to the patient (synergetic action), cost reduction, and long term treatments with reduced adverse effects.

Comparative study of transfer factor and acyclovir in the treatment of herpes zoster.

Reactivation of varicella herpes virus (VHV), latent in individuals who have previously suffered varicella, gives rise to herpes zoster and in some cases leads to a sequela of post herpetic neuritis with severe pain which is refractory to analgesics. Many different antiviral agents have been tried without achieving satisfactory results. Of all the antiviral agents employed, acyclovir has been the most successful in reducing post herpetic pain. However acyclovir has not been as reliable as interferon alpha (IFN-alpha). We have previously looked into the use of transfer factor (TF) as a modulator of the immune system, specifically with respect to its effectiveness in the treatment of herpes zoster. In this work findings from a comparative clinical evaluation are presented. A double blind clinical trial of TF vs acyclovir was carried out in which 28 patients, presenting acute stage herpes zoster, were randomly assigned to either treatment group. Treatment was administered for seven days and the patients were subsequently submitted to daily clinical observation for an additional 14 days. An analogue visual scale was implemented in order to record pain and thereby served as the clinical parameter for scoring results. The group treated with TF was found to have a more favorable clinical course, P < or = 0.015. Laboratory tests to assess the immune profile of the patients were performed two days prior and 14 days after initial treatment. The results of these tests showed an increase in IFN-gamma levels, augmentation in the CD4+ cell population but not the percentage of T rosettes in the TF treated group. These parameters were however insignificantly modified in patients receiving acyclovir. Although TF treated patients showed an increase in CD4+ counts these cells remained below the levels for healthy individuals. The fact that IFN-gamma levels as well as the counts for CD4+ cells rose in the TF treated group and not in the acyclovir one is very significant and confirms the immunomodulating properties of TF.

IgG antibody subclasses, tumor necrosis factor and IFN-gamma levels in patients with type II lepra reaction on thalidomide treatment.

A group of 9 Mexican lepromatous leprosy patients was studied at the beginning of a type II reaction (erythema nodosum leprosum, ENL) and after 1 or 2 months of thalidomide treatment. ENL patients at the onset of the reaction had slightly higher amounts of anti-Mycobacterium leprae IgG1 and IgG2 antibodies, compared to similar lepromatous patients that did not develop ENL. Neither these antibody levels nor IgM and the other IgG subclasses were importantly modified after thalidomide treatment. Serum TNF was significantly higher in the patients that developed ENL compared to those that did not develop the reaction. TNF levels were slightly decreased after 1 month of thalidomide treatment and significantly decreased after 2 months of treatment. Serum IFN-gamma was significantly lower in patients at the onset of ENL and was increased after 1 and 2 months of thalidomide treatment.