The dichotomy of the systemic and local cytokine responses of mucosal leishmaniasis patients in Sudan: A pilot study.

Sudanese mucosal leishmaniasis (ML) is a rare clinical form of leishmaniasis and characterized by persistent ulcer of the oral and/or the nasal mucous membranes caused by Leishmania donovani. No data is available about the systemic and local immune responses in mucosal leishmaniasis. This study aimed to measure the systemic and the local cytokines responses of Sudanese ML patients compared to cured cutaneous leishmaniasis patients (Leishmanin skin test positive, LST+ve) and unexposed healthy controls (Leishmanin skin test negative, LST-ve). Six parasitological confirmed ML patients, 7 LST+ve, and 6 LST-ve were enrolled. Systemic Th-1 (IFN-γ and TNF-α), Th-2 (IL-10 and IL-13), Treg (TGF-ß1), and inflammatory cytokines IL-6 and IL-8 concentration were measured in the supernatant of whole blood samples following stimulation with live L. donovani promastigotes using ELISA. Local intralesion IL-10, IFN-γ, and IL-13 expression was measured using Real Time PCR. A significant high concentrations of IFN-γ, TNFα, IL-10, TGFß, IL-6, and IL-8 were detected in the supernatant of stimulated whole blood samples of ML patients compared with the LST+ve and LST-ve controls. Using Real Time-PCR and primers for various cytokines, a significant high expression of TH2 cytokines IL-10 and IL-13 mRNA was detected in contrast to a low TH1 cytokine IFN-γ mRNA in the mucosal lesion. There is a clear dichotomy in the cytokine response during Mucosal leishmaniasis. A significantly high TH1, inflammatory and Treg cytokines response is produced systemically, in contrast to a significant high TH2 cytokines response in the mucosal lesion.

The dichotomy of the systemic and local cytokine responses of mucosal leishmaniasis patients in Sudan: A pilot study

@#Sudanese mucosal leishmaniasis (ML) is a rare clinical form of leishmaniasis and characterized by persistent ulcer of the oral and/or the nasal mucous membranes caused by Leishmania donovani. No data is available about the systemic and local immune responses in mucosal leishmaniasis. This study aimed to measure the systemic and the local cytokines responses of Sudanese ML patients compared to cured cutaneous leishmaniasis patients (Leishmanin skin test positive, LST+ve) and unexposed healthy controls (Leishmanin skin test negative, LST-ve). Six parasitological confirmed ML patients, 7 LST+ve, and 6 LST-ve were enrolled. Systemic Th-1 (IFN-γ and TNF-α), Th-2 (IL-10 and IL-13), Treg (TGF-β1), and inflammatory cytokines IL-6 and IL-8 concentration were measured in the supernatant of whole blood samples following stimulation with live L. donovani promastigotes using ELISA. Local intralesion IL-10, IFN-γ, and IL-13 expression was measured using Real Time PCR. A significant high concentrations of IFN-γ, TNFα, IL-10, TGFβ, IL-6, and IL-8 were detected in the supernatant of stimulated whole blood samples of ML patients compared with the LST+ve and LST-ve controls. Using Real Time-PCR and primers for various cytokines, a significant high expression of TH2 cytokines IL-10 and IL-13 mRNA was detected in contrast to a low TH1 cytokine IFN-γ mRNA in the mucosal lesion. There is a clear dichotomy in the cytokine response during Mucosal leishmaniasis. A significantly high TH1, inflammatory and Treg cytokines response is produced systemically, in contrast to a significant high TH2 cytokines response in the mucosal lesion.

Expression profiling of Sudanese visceral leishmaniasis patients pre- and post-treatment with sodium stibogluconate.

Visceral leishmaniasis (VL) in Sudan caused by Leishmania donovani is fatal in susceptible individuals if untreated. Treatment with sodium stibogluconate (SSG) leads to post-kala-azar dermal leishmaniasis (PKDL) in 58% of patients. Here, Affymetrix microarrays were used to identify genes differentially expressed in lymph nodes (N=9 paired samples) pre- and post-treatment with SSG. Using the Bioconductor package limma, 438 genes from 28 869 post-quality-control probe sets were differentially expressed (Pnominal ≤.02) post- vs pretreatment. Canonical pathway analysis using Ingenuity Pathway Analysis™ identified "role of nuclear factor of activated T-cell in regulation of immune response" (Pnominal =1.35×10-5 ; PBH-adjusted =4.79×10-3 ), "B-cell development" (Pnominal =2.04×10-4 ; PBH-adjusted =.024), "Fcγ receptor-mediated phagocytosis in macrophages and monocytes" (Pnominal =2.04×10-4 ; PBH-adjusted =.024) and "OX40 signalling" (Pnominal =2.82×10-4 ; PBH-adjusted =.025) as pathways differentially regulated post- vs pretreatment. Major network hub genes included TP53, FN1, MYC, BCL2, JUN, SYK, RUNX2, MMP1 and ACTA2. Top endogenous upstream regulators included IL-7 (P=2.28×10-6 ), TNF (P=4.26×10-6 ), Amyloid Precursor Protein (P=4.23×10-5 ) and SPI1/PI.1 (P=1.17×10-7 ). Top predicted chemical drug regulators included the flavonoid genistein (P=4.56×10-7 ) and the quinoline alkaloid camptothecin (P=5.14×10-5 ). These results contribute to our understanding of immunopathology associated with VL and response to SSG treatment. Further replication could identify novel therapeutic strategies that improve on SSG treatment and reduce the likelihood of progression to PKDL.

The 5q31 region in two African populations as a facet of natural selection by infectious diseases.

Cases of extreme natural selection could lead either to rapid fixation or extinction of alleles depending on the population structure and size. It may also manifest in excess of heterozygosity and the locus concerned will be displaying such drastic features of allele change. We suspect the 5q31 in chromosome 5 to mirror situation of such extreme natural selection particularly that the region encompasses genes of type 2 cytokine known to associate with a number of infectious and non-infectious diseases. We typed two sets of single nucleotide polymorphisms (SNPS) in two populations: an initial limited set of only 4 SNP within the genes of IL-4, IL-13, IL-5 and IL-9 in 108 unrelated individuals and a replicating set of 14 SN P in 924 individuals from the same populations with disregard to relatedness. The results suggest the 5q31 area to be under intense selective pressure as indicated by marked heterozygosity independent of Linkage Disequilibrium (LD); difference in heterozygosity, allele, and haplotype frequencies between generations and departure from Hardy-Weinberg expectations (DHWE). The study area is endemic for several infectious diseases including malaria and visceral leishmaniasis (VL). Malaria caused by Plasmodiumfalciparum, however, occurs mostly with mild clinical symptoms in all ages, which makes it unlikely to account for these indices. The strong selection signals seems to emanate from recent outbreaks of VL which affected both populations to varying extent.

Blindness following visceral leishmaniasis: a neglected post-kala-azar complication.

Visceral leishmaniasis (VL) is an important cause of morbidity and mortality that affects multiple organs. Post-kala-azar ocular involvement is a serious complication that can manifest as blepharo-conjuctivitis or pan-uveitis. Failure of prompt diagnosis and treatment can result in blindness. We report five cases with pan-uveitis that followed the successful treatment of VL and consequent post-kala-azar dermal leishmaniasis were presented. Two patients lost their sight permanently but the rest were successfully treated. A high index of suspicion and prompt treatment are of paramount importance in order to avoid blindness following post-kala-azar ocular uveitis.

Interleukin 10 gene polymorphisms and development of post kala-azar dermal leishmaniasis in a selected sudanese population.

BACKGROUND: Post kala-azar dermal leishmaniasis (PKDL) is a cutaneous form of disease that develops at variable times after individuals have received treatment for clinical visceral leishmaniasis (VL). The study aimed to investigate the possible role of interleukin 10 (IL-10) and development of PKDL. METHODS: 77 families composed of 41 complete case-parent trios and 36 case-parent pairs from the Masalit ethnic group were genotyped for 3 IL10 promoter polymorphisms: -1082A/G, -819C/T and -592C/A. RESULTS: Single point analysis using the transmission disequilibrium test showed no evidence of association between any of these IL10 promoter single nucleotide polymorphisms (SNPs) and development of PKDL. Haplotype analysis performed using TRANSMIT showed borderline significance between PKDL and the haplotype AA across -592C/A and -1082A/G (p = 0.053). Haplotypes GCC (0.33) and ATA (0.30) were the common haplotypes in this Sudanese population. Allele frequencies for the 3 SNPs differed significantly in Sudan compared to other African (Gambian, Malawian, YRI) populations. CONCLUSION: There is no evidence for an association between 3 SNPs in the IL10 gene promoter and susceptibility to PKDL in the Masalit ethnic group in Sudan, although some evidence for haplotype association was observed.

IFNG and IFNGR1 gene polymorphisms and susceptibility to post-kala-azar dermal leishmaniasis in Sudan.

Post-kala-azar dermal leishmanaisis (PKDL) in Sudan is associated with elevated interferon-gamma (IFN-gamma). To study interferon-gamma pathways in PKDL, we genotyped 80 trios from the Masalit ethnic group for polymorphisms at -470 ins/delTT, -270T/C, -56T/C and +95T/C in IFNGR1 and at -179G/A and +874T/A in IFNG. No associations occurred at IFNG. Global association with haplotypes comprising all four markers at IFNGR1 (chi(2)(10df)=21.97, P=0.015) was observed, associated with a significant (chi(2)(1df)=4.54, P=0.033) bias in transmission of the haplotype insTT T T T and less (chi(2)(1df)=5.59, P=0.018) than expected transmission of insTT C C C. When compared with data on malaria associations from Gambia, the results suggest a complex pattern of haplotypic variation at the IFNGR1 promoter locus associated with different infectious disease in African populations that reflect the complex roles of IFN-gamma in parasite killing versus inflammation and pathogenesis.

Dichotomy of protective cellular immune responses to human visceral leishmaniasis.

Healing/protective responses in human visceral leishmaniasis (VL) are associated with stimulation/production of Th1 cytokines, such as interferon IFN-gamma, and conversion in the leishmanin skin test (LST). Such responses were studied for 90 days in 44 adult healthy volunteers from VL non-endemic areas, with no past history of VL/cutaneous leishmaniasis (CL) and LST non-reactivity following injection with one of four doses of Alum-precipitated autoclaved Leishmania major (Alum/ALM) +/- bacille Calmette-Guerin (BCG), a VL candidate vaccine. The vaccine was well tolerated with minimal localized side-effects and without an increase in antileishmanial antibodies or interleukin (IL)-5. Five volunteers (5/44; 11.4%) had significant IFN-gamma production by peripheral blood mononuclear cells (PBMCs) in response to Leishmania antigens in their prevaccination samples (P = 0.001) but were LST non-reactive. On day 45, more than half the volunteers (26/44; 59.0%) had significantly high LST indurations (mean 9.2 +/- 2.7 mm) and high IFN-gamma levels (mean 1008 +/- 395; median 1247 pg/ml). Five volunteers had significant L. donovani antigen-induced IFN-gamma production (mean 873 +/- 290; median 902; P = 0.001), but were non-reactive in LST. An additional five volunteers (5/44; 11.4%) had low IFN-gamma levels (mean 110 +/- 124 pg/ml; median 80) and were non-reactive in LST (induration = 00 mm). The remaining eight volunteers had low IFN-gamma levels, but significant LST induration (mean 10 +/- 2.9 mm; median 11). By day 90 the majority of volunteers (27/44; 61.4%) had significant LST induration (mean 10.8 +/- 9.9 mm; P < 0.001), but low levels of L. donovani antigen-induced IFN-gamma (mean 66.0 +/- 62 pg/ml; P > 0.05). Eleven volunteers (11/44; 25%) had significantly high levels of IFN-gamma and LST induration, while five volunteers had low levels of IFN-gamma (<100 pg/ml) and no LST reactivity (00 mm). One volunteer was lost to follow-up. In conclusion, it is hypothesized that cellular immune responses to human VL are dichotomatous, and that IFN-gamma production and the LST response are not in a causal relationship. Following vaccination and probably cure of VL infection, the IFN-gamma response declines with time while the LST response persists. LST is a simple test that can be used to assess candidate vaccine efficacy.

Diagnosis of tuberculous lymphadenitis by FNAC, microbiological methods and PCR: a comparative study.

Despite its usefulness in the diagnosis of tuberculous lymphadenitis, fine needle aspiration cytology (FNAC) faces several limitations, and its sensitivity and specificity are not well established. The diagnostic accuracy and limitations of FNAC were studied in comparison with conventional microbiological methods and polymerase chain reaction (PCR). Sixty patients with lymphadenopathy and a clinical diagnosis of tuberculous lymphadenitis were subjected to FNA. The aspirate was used for cytological examination, Ziehl-Neelsen staining, mycobacterial culture and PCR. PCR was performed using two sets of oligonucleotide primers for Mycobacterium tuberculosis and a single primer for M. bovis species. The results of FNAC, microbiological methods and PCR correlated with the clinical outcome after follow-up for an average period of 24 months. Twenty-five cases (41.6%) were treated and responded well to anti-tuberculosis therapy, among them 17 were correctly diagnosed by FNAC (68%), eight by microbiological methods (32%) and 24 by PCR (96%). When PCR is considered the gold standard, FNAC predicted the correct diagnosis in 62% of cases with a high false negative rate (38%) due to the absence of granuloma/necrosis in smears from cases of early tuberculosis. In the latter group PCR proved to be the most valuable and a diagnostic success of 100% was achieved when FNAC and PCR were combined. In addition, PCR allowed immediate characterization of M. tuberculosis in the vast majority (96.2%) of cases in the study population.

Adjuvant-induced arthritis: IL-1 beta, IL-6 and TNF-alpha are up-regulated in the spinal cord.

Adjuvant-induced arthritis (AIA) is a widely used animal model of human rheumatoid arthritis (RA). We have previously shown that increased neuropeptide expression is observed in the spinal cord of AIA rats. To study the potential role of cytokines in the spinal cord of AIA, we wanted to determine whether there are changes of glial and cytokine expression (IL-1 beta, IL-6, TNF-alpha and IFN-gamma) in the spinal cord of AIA rats. Our data indicated that macroglia and MHC class II immunostaining were enhanced, astrocytes expressing GFAP were increased in number and immunostaining intensity. Using in situ hybridization and immunohistochemical methods, both mRNA and protein levels of IL-1 beta, IL-6 and TNF-alpha were significantly increased in the spinal cord of arthritic rats. Increased cytokine expression was presented in the reactive astrocytes and microglia.

Effects of short- and long-term rat hind limb immobilization on spinal cord insulin-like growth factor-I and its receptor.

In this study we investigated changes in the spinal cord insulin-like growth factor-I peptide (IGF-I) and its receptors (IGF-IR) after hind limb immobilization for 5 days, 2, 4, and 8 weeks. Moreover, effects on IGF-I and nicotinic cholinergic receptors (nAChRs) in two types of skeletal muscle were also investigated. IGF-I levels were measured by radioimmunoassay (RIA) whereas IGF-IR and nAChRs were measured by quantitative receptor autoradiography. Spinal cord IGF-I levels decreased significantly after 5 days, 2 and 4 weeks of immobilization, whereas IGF-IR increased significantly after 4 and 8 weeks compared to controls. In skeletal muscles, nAChRs increased significantly after 5 days and 2 weeks in the soleus (SOL) and tibialis anterior (TIB) muscles, respectively, and continued up to 8 weeks in both muscles. IGF-I concentration decrease significantly after 4 and 8 weeks in the SOL and TIB muscles, respectively. Despite the normal levels of IGF-I in both muscles at the early time points (5 days and 2 weeks), low levels of IGF-I were observed concurrently in the spinal cord ipsilateral to the immobilized limb. Our findings suggest that the early decrease in the IGF-I level and the late upregulation in the IGF-IR in the spinal cord might represent a nervous system response to disuse.

Increased levels of somatostatin in rat ankles with adjuvant arthritis.

Levels of somatostatin were investigated in the ankles and spinal cords of rats suffering from acute and chronic adjuvant arthritis. As measured by radioimmunoassay, somatostatin showed significantly higher concentrations only in chronic arthritic ankles. No significant difference was observed in somatostatin levels between the spinal cords of normal and arthritic groups. Using immunohistochemical labeling and electron microscopy, we observed increased somatostatin labeling in the mature bone matrix, monocytes, and polymorphonuclear cells of bone marrow and macrophage-like synovial cells of chronically arthritic rats. Understanding the mechanism(s) which lead to increased somatostatin in chronic arthritic joints may result in more effective treatment methods.

Changes in tissue levels of growth hormone, insulin-like growth factor-I, and somatostatin in the femurs of hind-limb immobilized rats.

Immobilization of an extremity causes skeletal muscle atrophy and a dramatic increase in bone resorption. Growth hormone (GH) is known to play an important role in bone remodeling mediated in part by local insulin-like growth factor-I (IGF-I). In this study, we investigated changes in the levels of GH and IGF-I peptide in bone extracts from the femur after hind-limb immobilization for 5 days, 2, 4, and 8 weeks. The levels of somatostatin, which interacts with GH, were also measured in the bone extracts. GH levels increased after 8 weeks of hind-limb immobilization whereas the IGF-I concentrations increased after 2 weeks, but returned to control levels at 4 weeks, and decreased after 8 weeks of immobilization. The somatostatin levels in the bone extracts increased only after 8 weeks of hind-limb immobilization. Our findings suggest that, after hind-limb immobilization, changes in the concentrations of GH, IGF-I, and somatostatin in bone may mediate bone resorption either directly or through interaction with other factors.

Suppression of autoimmune neuritis in IFN-gamma receptor-deficient mice.

Experimental autoimmune neuritis (EAN) is an animal model of the human disease Guillain-Barré syndrome. In this autoimmune inflammatory disease, CD4(+) T cells mediate demyelination in the peripheral nervous system (PNS). Infiltrating macrophages and T cells as well as cytokines like interferon (IFN)-gamma are intimately involved in causing pathogenic effects. To investigate the role of IFN-gamma in cell-mediated EAN, IFN-gamma receptor-deficient mutant (IFN-gammaR(-/-)) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180-199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. IFN-gammaR(-/-) mice exhibited later onset of clinical disease. The disease was also less severe than in wild-type mice. Fewer IL-12-producing but more IL-4-producing cells were found in sciatic nerve sections from IFN-gammaR(-/-) mice than from wild-type mice on day 24 postimmunization, i.e., at the peak of clinical EAN. At the same time, IFN-gammaR(-/-) mice had less infiltration of inflammatory cells, including macrophages, CD4(+) T cells, and monocytes, into sciatic nerve tissue and less demyelination. However, numbers of IFN-gamma-secreting cells from the spleen were significantly augmented in the IFN-gammaR(-/-) mice, reflecting a failure of negative feedback circuits. The IFN-gammaR deficiency did not affect the production of anti-P0 peptide 180-199-specific antibodies. These results indicate that IFN-gamma contributes to a susceptibility for EAN in C57BL/6 mice by promoting a Th1 cell-mediated immune response and suppressing a Th2 response.

Intracerebroventricular administration of somatostatin prevents and attenuates adjuvant arthritis.

The effects of somatostatin on the development of adjuvant arthritis induced by Mycobacterium butyricum were studied. Somatostatin was injected into the lateral cerebral ventricle every day for 14 days beginning on the first day of mycobacteria inoculation in the preventive group. In the treatment group, somatostatin was injected from day 17 until day 30 post-mycobacteria inoculation. Arthritis was evaluated by measuring ankle joint circumference and diameter as well as microscopic examination of ankle joint sections. Somatostatin profoundly inhibited the development of adjuvant arthritis and an anti-inflammatory action was observed in the treatment group. These results suggest that somatostatin has a central action that can prevent or attenuate symptoms associated with arthritis.

CD28-B7 costimulation: a critical role for initiation and development of experimental autoimmune neuritis in C57BL/6 mice.

CD28 provides a critical costimulatory signal for antigen-specific T cell activation. Because CD28 is an important factor in the development of autoimmune diseases, we investigated its role in T cell-mediated experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in humans. CD28-deficient mutant (CD28-/-) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180-199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. As a result, all wild-type mice developed severe EAN, in contrast, none of the CD28-/- mice manifested clinical signs of disease. Additionally, CD28-/- mice had fewer IL-12 producing cells in sciatic nerve sections and fewer IFN-gamma secreting splenic cells than wild-type mice on day 24 post immunization, i.e., at the peak of clinical EAN. At that time point, CD28-/- mice had milder infiltration of such inflammatory cells as macrophages, CD4+ T cells and monocytes into sciatic nerve tissues and less demyelination than wild-type mice. Moreover, the CD28-deficiency led to reduced production of specific anti-P0 peptide 180-199 antibodies compared with wild-type mice. Evidently, CD28 is required for interaction with B7 to regulate the activation of T and B cells that initiates development of EAN.

Intracerebroventricular met-enkephalin administration modulates adjuvant arthritis.

The purpose of this study was to investigate the anti-inflammatory properties of intracerebroventricular met-enkephalin (met-enk) administration in an animal model of arthritis. Adjuvant arthritis was induced in rats by intradermal inoculation of mycobacterium butyricum and the effects of intraventricular met-enk+thiorphan (enkephalinase inhibitor) were studied. Treatment was initiated either simultaneously with the bacterial inoculation (preventive group) or on post-inoculation day 17 after the appearance of inflammation (treatment group). The degree of inflammation was evaluated by measuring the diameter and the circumference of the ankle joint immediately before the sacrifice (day 31) and by histologic examination of ankle joint sections. The results of this study revealed that combined intraventricular injections of met-enk+thiorphan reduced the arthritic-like inflammation in the preventive group as well as in the treatment group. These findings suggest that centrally applied met-enk+thiorphan may suppress the development adjuvant arthritis as well as the symptoms of manifest arthritis. Thus central met-enk may be involved in both hypothalamic pituitary adrenal axis and immune forms of stress-induced modulation.

Neuromuscular effects of two types of fracture treatment.

Immobilisation causes denervation-like changes in the motor endplates, decreases the content of IGF-I, and increases the number of IGF-I receptors in the spinal cord. In the rat we investigated whether similar changes occur after a fracture of the midshaft of the femur which had been treated by intramedullary fixation with adequate or undersized pins. A more pronounced reduction in muscle wet weight was seen after fixation by undersized pins as well as decreased ash density of the ipsilateral tibia which did not completely return to normal within the 12-week experimental period. The nicotinic cholinergic receptors in the motor endplates of tibialis anterior were increased (p < 0.01) and there was a significant increase (p < 0.02) in IGF-I receptors in the lumbar spinal cord ipsilateral to the fracture after treatment by undersized nails. These changes may be associated with the impaired proprioception, co-ordination and motor activity which are sometimes seen after fractures.

High levels of C-reactive protein in the peripheral blood during visceral leishmaniasis predict subsequent development of post kala-azar dermal leishmaniasis.

Post kala-azar dermal leishmaniasis (PKDL) is a known sequel to visceral leishmaniasis in India and East Africa, and in Sudan about 50% of the kala-azar patients develop PKDL. In this study we followed kala-azar patients from diagnosis and up to 2 years after initiation of treatment. During the first 6 months some developed PKDL (group 1), while some did not develop PKDL (group 2). We measured the plasma levels of C-reactive protein (CRP) at diagnosis of kala-azar (day 0), during treatment (day 15), after treatment (day 30) and later during the follow up period. At day 0, plasma CRP levels were higher in patients who later developed PKDL (group 1) than in patients who did not develop PKDL subsequently (group 2) (P = 0.008). At days 15 and 30, the CRP levels were comparable in the two groups, and lower than at day 0. We have previously shown that high plasma levels of IL 10 and in keratinocytes during visceral leishmaniasis predict subsequent development of PKDL. The method however requires expensive equipment and reagents. The results of the present study indicate that kala-azar patients, who have a high risk of developing PKDL after treatment can be identified by measuring plasma CRP.