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.

Cutaneous leishmaniasis caused by members of Leishmania braziliensis complex in Nayarit, State of Mexico.

An epidemiological study was carried out in the northern Mexican state, Nayarit. Fourteen patients with possible cutaneous leishmaniasis skin lesions gave positive Montenegro skin tests. Biopsies were taken from the skin ulcer and analyzed by polymerase chain reaction (PCR) with specific primers for the Leishmania mexicana complex; however all biopsies were not amplified. PCR carried out with specific primers for the L. braziliensis complex resulted in the amplification of all patient DNA. DNA from 12 out of 14 biopsies gave positive amplification with primers species specific for L. (Viannia) braziliensis and hybridized with a species specific L. (V.) braziliensis probe. These results demonstrate the presence in Nayarit of at least two members of the L. braziliensis complex. Most of the cutaneous lesions were caused by L. (V.) braziliensis and two by another species belonging to the L. braziliensis complex. As far as we are aware, this is the first report of L. (V.) braziliensis in Nayarit. The main risk factor associated with the contraction of this disease in Nayarit is attributed to working on coffee plantations.

Visceral leishmaniosis caused by Leishmania (L.) mexicana in a Mexican patient with human immunodeficiency virus infection.

A 36 year old male was admitted in December 1997 to hospital with afternoon fever, malaise and hepatosplenomegaly. He also had a dry cough, dyspnoea and anaemia. Pneumonia caused by Pneumocystis carinii and human immunodeficiency virus (HIV) infection were documented. The HIV infection was confirmed in 1997 with 290,000 virus copies. The patient had been in the Mexican State of Chiapas which is known to be endemic for visceral leishmaniosis (VL) and localized cutaneous leishmaniosis (LCL). The visceral symptoms were diagnosed as VL and the causal agent was identified as Leishmania (L. ) mexicana. Identification of Leishmania was carried out by the analysis of amplified DNA with specific primers belonging to the Leishmania subgenus and by dot blot positive hybridisation of these polymerase chain reaction derived products with kDNA from the L. (L. ) mexicana MC strain used as probe. This is the first case in Mexico of VL caused by a species of Leishmania that typically produces a cutaneous disease form.

Aetiology of visceral leishmaniasis in Mexico.

Two children with visceral leishmaniasis (VL), were studied by DNA analysis. DNA from liver biopsy samples from both patients, was amplified by PCR with broad primers specific for the Leishmania subgenus. DNA from the patient from Chiapas was also amplified with primers specific for the Leismania donovani complex and hybridised with a probe specific for L. donovani complex. The second patient, who is the first reported case of visceral leishmaniasis in the Mexican state of Tabasco, where localised cutaneous leishmaniasis and DCL predominate, had a co-infection with Toxoplasma gondii. The DNA from this patient was not amplified with primers specific for the L. donovani complex, did not hybridise with a probe specific for the L. donovani complex, but did hybridise with kDNA from a Mexican Leishmania mexicana strain used as a probe. We therefore, suggest that members of the L. donovani or L. mexicana complexes cause VL in Mexico.

Plasmodium chabaudi chabaudi: effect of low parasitemias on immunity in CB6F1 mice.

We examined the effect that low parasitemias have on the immune response of CB6F1 mice infected with Plasmodium chabaudi chabaudi AS. Ascending parasitemias were stopped by chloroquine treatment when they were between 1.6 and 9.4%. Mice that suffered low parasitemias developed good immunity to homologous reinfection but, contrary to what happened in mice that suffered full parasitemias, they did not develop immunity to heterologous reinfection with Plasmodium yoelii 17XL. Total IgG antiparasite antibody responses were similar in mice that suffered low or full parasitemia, both in primary infection and after reinfection. At the level of isotypes, IgM, IgG1, IgG2b, and IgG3 responses were similar in mice that suffered low or full parasitemias, but after reinfection, mice that suffered low parasitemias responded with higher levels of IgG2a than mice that suffered full parasitemias. Mice that suffered low parasitemias did not have splenomegaly but their immunity to homologous reinfection was diminished after splenectomy in a manner similar to that of splenectomized mice that suffered full parasitemia. CB6F1 mice can develop homologous immunity even if exposed to low parasitemias but cannot develop heterologous immunity unless exposed to high parasite loads.

Identification of Mexican Leishmania species by analysis of PCR amplified DNA.

Leishmania parasites isolated into culture from patients with LCL or DCL from four different Mexican states were characterised using polymerase chain reaction (PCR), hybridisation with specific probes, and isoenzymes. PCR of the parasites showed that 10 of 11 of those isolates were members of the mexicana complex. This was confirmed in seven cases by isoenzymes. Restriction enzyme digests of PCR products of Mexican isolates showed the isolates to be different from the L.(L.) mexicana reference strain BEL21. Two (C2 and AM) of the isolates were shown to be a possible mixed infection between mexicana and braziliensis complex members. With a second set of samples from different patients from Campeche state, PCR of 14 biopsies indicated the presence of braziliensis complex members in six of the samples. The results showed that most of our isolates of Leishmania which come from the states of Tabasco and Veracruz are members of the Leishmania mexicana complex, but they seem to be different from the L.(L.) mexicana BEL21 reference strain. By hybridisation most of the biopsies (seven out of 14) from Campeche belong to the L. braziliensis complex and two out of 14 to L. mexicana complex and three out of 14 hybridised with both complexes, and two biopsies were negative. In Campeche, which is very close to Tabasco state and has border with Guatemala, we found members of the L. mexicana and L. braziliensis complexes.

The spleen, IgG antibody subsets and immunity to Plasmodium berghei in rats.

The development of IgG subclass-specific antibody responses to Plasmodium berghei in spleen-chimeric rats were monitored to determine if there was any relationship between IgG subset profiles and resistance. Strongly immune eusplenic rats respond to challenge with P. berghei by producing high levels of parasite-specific IgG2a, IgG2b and IgG2c but only modest levels of IgG1. Splenectomy profoundly affects the antibody response to infection. Thus, in splenectomized immunized rats, which harbour a chronic parasitaemia of 1%, the IgG2a, IgG2b and IgG2c responses peak 1 week later than in eusplenic immunized rats although the size of the peak is similar. More marked effects are apparent in the IgG1 response, the magnitude of which is far greater in splenectomized immunized rats than eusplenic immunized rats. Similar antibody profiles are seen in splenectomized immunized rats transplanted with a naive spleen. In contrast, splenectomized naive rats receiving either a transplant of a spleen from an immune rat or a transfer of immune spleen cells have high levels of IgG2a, IgG2b and IgG2c but modest levels of IgG1. However, only the former group of rats completely clears the parasite, the latter maintaining a chronic 1% parasitaemia. Thus, although complete resistance to P. berghei is always associated with high levels of parasite-specific IgG2a, IgG2b and IgG2c plus modest levels of IgG1, this is not a sufficient set of conditions to guarantee complete immunity. The IgG subset profile may be related to cytokine production; IFN-gamma was detected in the sera of rats receiving spleens from rats immune to P. berghei (modest IgG1 responses) but not in rats receiving spleens from naive animals (pronounced IgG1 responses).

Seroepidemiological studies of cutaneous leishmaniasis in the Campeche state of México.

Seroepidemiological studies of cutaneous leishmaniasis were carried out in 169 individuals in a rural area of the Campeche state of México. Fifty showed cutaneous lesions suggestive of leishmaniasis, 70% were parasite positive and 96% skin test positive. An overall 40% positivity to skin test with Montenegro's antigen was found. Most of the affected individuals were males from 11 to 30 years-old. Antibodies were determined by immunofluorescent antibody test (IFA) and by Western blot. Two antigen preparations were used, one from a Leishmania mexicana strain which produced localized cutaneous leishmaniasis (LCL) and the other from a diffuse cutaneous leishmaniasis (DCL). In the general population from the area of study 19% gave positive IFA tests with DCL antigen and 20% with LCL antigen while for the patients 67% gave positive IFA tests with DCL and 71% with LCL. By Western blot analysis most of the patients recognized more antigens in the DCL than in the LCL strain. In the DCL strain 78% of patients recognized a 105 kDa, 34% a 139 kDa, 28% a 117 kDa and 26% a 205 kDa MW antigen. In the LCL strain 40% of patients recognized a 205 kDa and 22% a 175 kDa antigens.

Protection of rats against malaria by a transplanted immune spleen.

A number of reports have suggested that the spleen plays a key role in the regulation of immunity to malaria but the role, if any, of other tissues is less clear. Furthermore, numerous functional changes occur in the spleen following malaria infection and it is not known whether the spleen's role relates primarily to its content of malaria-specific lymphocytes or to the altered structure and function that has occurred. To address these issues we have generated splenic chimeras by transplanting spleens between Plasmodium berghei-immune and naive rats. In the absence of a functional spleen, specific immune responses from both isolated splenic and non-splenic cells can partially control infection. However, an immune spleen in a naive rat can solidly protect the animal from malaria and a normal spleen in an otherwise immune rat can provide enhanced protection over the non-splenic state. Thus, in the presence of functional splenic architecture both splenic and non-splenic malaria-specific lymphocytes operate more effectively. However, these studies do demonstrate an important role for non-splenic tissue in immunity at least for P. berghei in the rat. The study could have important implications for induction of protective immune responses by vaccination and suggests that malaria-specific lymphocyte responses induced in the periphery following vaccination could interact with parasites in both spleen-dependent and spleen-independent ways.

Infection of BALB/c, C57B1/6 mice and F1 hybrid CB6F1 mice with strains of Leishmania mexicana isolated from Mexican patients with localized or diffuse cutaneous leishmaniasis.

Mice from the syngeneic strains BALB/c, C57B1/6 and (BALB/cxC57B1/6)F1 hybrids (CB6F1) were infected in the footpad with six different strains of Leishmania mexicana mexicana isolated from Mexican patients. Three Leishmania strains were isolated from patients with localized cutaneous leishmaniasis (LCL, the benign form of the disease) and three from patients with diffuse cutaneous leishmaniasis (DCL, the malignant form of the disease). In BALB/c mice, four Leishmania strains showed a sustained fast growth from 4 to 5 weeks postinfection until the end of the experiment (15 weeks), and the other two grew slowly up to 10 or 12 weeks after infection and then started to grow faster. In C57B1/6 mice four Leishmania strains showed a limited to moderate growth up to 6 to 11 weeks postinfection and then started to decrease. One strain showed a moderate growth during the entire experiment and one strain grew as fast as in BALB/c mice up to 11 weeks postinfection and then started to decrease. The CB6F1 hybrid behaved like the C57B1/6 parent strain with five Leishmania strains but was much more resistant to one Leishmania strain than the C57B1/6 mice. Sex of the mouse did not influence the outcome of infection. One important purpose of this work was to see if the Leishmania strains that cause DCL are intrinsically more virulent than those that cause the benign form (LCL). Although important variations in virulence among the Leishmania strains were observed, especially in BALB/c mice, they were not correlated with the type of disease caused in humans.

Some studies on the experimental infection of golden hamsters with Taenia solium.

Golden hamsters were infected orally with viable cysticerci of Taenia solium obtained from infected pigs. After two weeks of infection implanted scolices of about 4 mm were found in exactly the same number as the number of ingested cysticerci. At six weeks 66% of the ingested cysticerci were found as implanted tapeworms (average size: 5.7 cm). At ten weeks 16% of the ingested cysticerci were found as implanted tapeworms (average size: 5.8 cm). At 14 weeks no tapeworms were found. Skin tests with taenia extracts were positive after 9 weeks of infection peaked at 12 and 14 weeks and declined afterwards becoming negative after 27 weeks. Skin test with cysticercus extracts were weaker, peaked at 8 and 10 weeks, were very low after 12 weeks and became negative after 16 weeks. Histological studies in the attachment site at the small intestine showed at 2 weeks a cellular infiltrate formed by macrophages, epithelioid cells and some plasma cells, there was very little alteration of epithelium. At 6 and 8 weeks the epithelium was damaged and necrotized. At 17 and 19 weeks the lesion started to resolve. We conclude that the golden hamster can be used to reproduce in the laboratory at least part of the life cycle of Taenia solium.

[Recognition by immunoelectroblotting of antigens from Taenia solium and its larva]. / Reconocimiento por inmunoelectrotransferencia de antígenos de Taenia solium y su larva.

Golden hamster (Mesocricetus auratus) were infected with cysticerci of Taenia solium. Groups of three were bled and killed weekly up to 15 weeks recording the number of implanted adults in the small intestine. A longitudinal study on the antibody response against adult and larvae Ags was carried out, as well as against Ags of H. nana. By ELISA, antibodies against larvae and adult Ags of T. solium were detected since the first week, showing a peak at 3 and 5 weeks respectively. At 14 weeks antibodies levels were very low, which is the time when the parasite is eliminated. The response against Ags of H. nana was very low and disappeared at 4 weeks. By western blot we found that the infected hamsters can distinguish specific stage Ags both in the adult and larvae of T. solium. After 12 weeks only the adult was recognized, the Ags of H. nana were recognize in western blot only during the first week.