Mucosal leishmaniasis: in situ characterization of the host inflammatory response,before and after treatment

Mucosal leishmaniasis (ML) generally shows progressive tissue destruction, not yet fully elucidated, associated with an intense inflammatory response. To contribute to the understanding of this process and of how treatment interferes with it, we studied several anatomopathological parameters, including those analyzed by immunohistochemistry, such as Leishmania antigens, cells participating in the immune response and cytokine expression. Biopsies were taken from 20 patients with ML before and after treatment. A mixed Th1 and Th2 pattern response occurred inside ML before treatment, persist after treatment. Nevertheless, this mixed response was smaller than in active lesions, with reduced but present numbers of cells expressing TNF-alpha, IFN-gamma and IL-4 and sustained numbers of cells expressing IL-10. We may conclude that specific treatment causes a reduction of inflammatory lesions and disappearance of amastigote forms of Leishmania although the factors related to the pathogenesis of the lesion, such as T CD4+ and T CD8+ lymphocytes and Leishmania antigens, persist in treated lesions. The maintenance of these inflammatory patterns may be due to a specific host-parasite relationship response, strongly indicating the need for continuous surveillance of LM patients at risk of reactivation, despite effective cicatrization after therapy.     

Differential immune regulation of activated T cells between cutaneous and mucosal leishmaniasis as a model for pathogenesis

Cutaneous (CL) and mucosal leishmaniasis (ML) are characterized by a predominant type 1 immune response (IFN-gamma and TNF-alpha production) and strong inflammatory response in the lesions with few parasites. This exacerbated type 1 response is more evident in ML as compared to CL. Our main hypothesis is that a differential immune regulation of T cell activation leads to over reactive T cells in ML. In the present study, we investigated immunological factors that could explain the mechanisms behind it by comparing some immune regulatory mechanisms between ML and CL patients: frequency of cells expressing co-stimulatory molecules, apoptotic markers, T cell activation markers; and ability of neutralizing antibodies to IL-2, IL-12 and IL-15 do down-regulate IFN-gamma production in leishmania antigen-stimulated peripheral blood mononuclear cells (PBMC). Interestingly, in CL anti-IL-2 and anti-IL-15 significantly suppressed antigen-specific IFN-gamma production, while in ML only anti-IL-2 suppressed IFN-gamma production. Finally, higher frequency of CD4+ T cells expressing CD28-, CD69+ and CD62L(low) were observed in ML as compared to CL. These data indicate that an exacerbated type 1 response in ML is differentially regulated and not appropriately down modulated, with increased frequencies of activated effectors T cells, maintaining the persistent inflammatory response and tissue damage observed in ML.     

Correlation between histopathology, immune response, clinical presentation, and evolution in Leishmania braziliensis infection

Skin biopsies from 221 parasitologically confirmed cases of tegumentary leishmaniasis caused by Leishmania braziliensis spp. were evaluated with respect to histopathology, the qualitative and quantitative nature of the cellular infiltrate, and the presence of Leishmania amastigotes. These variables were cross correlated with the Leishmania-specific immune response, clinical presentation, and response to treatment. Physical evidence of prior leishmanial lesions was associated with the absence of amastigotes (P less than or equal to 0.001) and the presence of giant (P = 0.03) and epitheloid cells (P = 0.03) in the biopsy of the active lesion. The presence of amastigotes was inversely related to the duration of the lesion (P less than or equal to 0.001) and the presence of eosinophils (P less than or equal to 0.01), whereas the presence of adenopathy (P = 0.01), necrosis (P = 0.001), histiocytes (P = 0.001), and increased serum antibody titer (P = 0.02) were directly associated with the presence of amastigotes. The lymphocyte transformation response was correlated with the presence of granulomas (P = 0.001), but showed no correlation with cutaneous delayed type hypersensitivity. The presence of epithelioid (P = 0.04) and giant cells (P = 0.03) was associated with less drug being required to achieve healing. In contrast, necrosis was associated with a greater amount of drug to achieve healing (P = 0.05). The observed correlations between tissue responses and immune and clinical parameters provide further evidence for the role of antibody and other soluble mediators of the cellular immune response in the evolution of disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased production of tumour necrosis factor-alpha interleukin-1 beta, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn's disease.

BACKGROUND: Increasing evidence points to a important role for inflammatory cytokines for the pathogenesis of Crohn's disease. AIM: To compare the secretion rate of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) by morphologically normal and inflamed intestinal mucosa from patients with Crohn's disease. RESULTS: Organ cultures of intestinal biopsy specimens taken from areas of affected mucosa from patients with Crohn's disease spontaneously produced increased amounts of TNF-alpha, IL-1 beta, and IL-6 compared with controls but also biopsy specimens taken in macroscopically and microscopically unaffected areas in the same patients. Concentrations of IL-1 beta and IL-6 measured in the supernatant fluid of biopsy cultures were positively correlated with the degree of tissue involvement measured by both endoscopic and histological grading. By contrast, TNF-alpha concentrations were not correlated to endoscopic and histological grading. CONCLUSIONS: These consistently raised TNF-alpha, IL-1 beta and IL-6 secretions by normal appearing mucosa from patients with Crohn's disease provide evidence for a sustained immune stimulation in Crohn's disease even in the absence of patent inflammation. The results shed a new light on the role of inflammatory cytokines in the onset of intestinal tissue damage in Crohn's disease and suggest that the range of intestinal lesions in Crohn's disease may be wider than suspected on the basis of regular endoscopic and histological examinations.     

Healing of chronic necrobiosis lipoidica lesions in a type 1 diabetic patient after pancreas-kidney transplantation: a case report

Necrobiosis lipoidica (NL) is a degenerative disease of dermal connective tissue of unknown etiology characterized by erythematous plaques preferentially localized to distal extremities. Skin lesions show a chronic relapsing nature. NL is often associated with diabetes mellitus and satisfactory treatment options are lacking. We describe the spontaneous healing of NL lesions after pancreas and kidney transplantation in a Type 1 diabetic patient with chronic NL recalcitrant to a variety of standard treatments. The 31-yr-old male patient had experienced NL lesions for more than 15 yr; despite various systemic and topical treatments, the skin lesions had pregressively enlarged. Because of end-stage renal disease, a simultaneous pancreas and kidney transplantation was performed and immunosuppressive therapy with tacrolimus (TAC), mycophenolate mofetil (MMF), and prednisone was started. Pancreatic transplantation maintained satisfactory metabolic control with no need of exogenous insulin. After transplantation, skin lesions slowly healed without any specific treatment, leaving residual areas of fibrotic scars. A skin biopsy confirmed the absence of typical NL lymphocytic and histiocytic inflammatory response. Clinical remission of NL lesions may probably be explained by the concomitant effect of multiple-drug regimen for immunosuppression (TAC, MMF, and prednisone) and improved skin microcirculation secondary to the good metabolic control provided by pancreas transplantation.     

Placebo controlled treatment of Ecuadorian cutaneous leishmaniasis.

Pentavalent antimony has been considered to be the standard treatment for leishmaniasis, but more recently, the orally administrable agent allopurinol ribonucleoside has been the subject of several clinical trials. In this study, these two agents were evaluated in patients with Ecuadorian cutaneous leishmaniasis. Patients were randomly assigned to the two treatment groups. The mean reduction in lesion size for the 28 patients treated with Pentostam (20 mg Sb/kg/day intramuscularly for 20 days) was 61%, 23%, and 11% after one, two, and three weeks, respectively. There was a wide range in the individual values, and some lesions markedly enlarged in the first week of therapy. An initially healed lesion was defined as one that had greater than 80% re-epithelialized by the 1.5-month post-treatment followup. All Pentostam patients demonstrated this degree of lesion resolution (100% initial healing rate), but one patient showed evidence of relapse at the three month followup resulting in a 96% complete healing rate for the 12 month observation period. Patients in the untreated control group demonstrated a strikingly high rate of healing with 9 of 12 patients having re-epithelialized all lesions after 1.5 months observation (75% initial healing rate). The mean reduction in lesion size for the untreated patients was 56%, 29%, and 25% after one, two, and three weeks, respectively. Twenty-one patients received allopurinol ribonucleoside (1,500 mg QID) plus probenecid (500 mg QID) for 28 days. Lesions in nine of these patients were healed at the time of the 1.5 month followup (41% healing rate).(ABSTRACT TRUNCATED AT 250 WORDS)     

Randomized, double-blind study of stibogluconate plus human granulocyte macrophage colony-stimulating factor versus stibogluconate alone in the treatment of cutaneous Leishmaniasis.

The response to recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) in the treatment of cutaneous leishmaniasis was evaluated. Twenty patients with cutaneous leishmaniasis who had lesions for 60 days were enrolled in a double-blind placebo trial of GM-CSF with standard parenteral sodium stibogluconate (20 mg/kg-1/day-1) for 20 days. Ten patients were randomized to receive intralesionally injected GM-CSF (200 microgram) at enrollment and 1 week after, and 10 patients received saline as placebo. GM-CSF- and antimony-treated patients healed faster than patients who received antimony alone (49+/-32.8 vs. 110+/-61.6 days, P<.05). Seven of 10 patients were healed of their lesions before 40 days after therapy in the GM-CSF group, compared with only 1 of 10 patients in the placebo group (relative risk, 7; 95% confidence interval, 1.04-47.00). Thus, GM-CSF plus antimony significantly increased the chance of lesion healing in 40 days.     

The wound repair response controls outcome to cutaneous leishmaniasis

Chronic microbial infections are associated with fibrotic and inflammatory reactions known as granulomas showing similarities to wound-healing and tissue repair processes. We have previously mapped three leishmaniasis susceptibility loci, designated lmr1, -2, and -3, which exert their effect independently of T cell immune responses. Here, we show that the wound repair response is critically important for the rapid cure in murine cutaneous leishmaniasis caused by Leishmania major. Mice congenic for leishmaniasis resistance loci, which cured their lesions more rapidly than their susceptible parents, also expressed differentially genes involved in tissue repair, laid down more ordered collagen fibers, and healed punch biopsy wounds more rapidly. Fibroblast monolayers from these mice repaired in vitro wounds faster, and this process was accelerated by supernatants from infected macrophages. Because these effects are independent of T cell-mediated immunity, we conclude that the rate of wound healing is likely to be an important component of innate immunity involved in resistance to cutaneous leishmaniasis.     

Healing of endoscopic biopsy sites in the human rectum

The time required in man for a rectal ulcer created with endoscopic biopsy forceps to heal is unknown. To answer this question, we created a large ulcer (approximately 8 mm in diameter) and a smaller ulcer (approximately 4 mm in diameter) in four healthy young men and followed the healing of the ulcers by visual examination every 4 days. Complete healing of the large ulcer had not occurred on days 4 and 8, while by day 12, three had healed and by day 16, all four had disappeared. Of the small ulcers, two had healed by day 8, and all had healed by day 12. This study suggests that it takes approximately 8-12 days for an artificially created rectal lesion to heal completely.     

Adult Onset of Multifocal Eosinophilic Granuloma of Bone: A Long-term Follow-up with Evaluation of Various Treatment Options and Spontaneous Healing

We report a case of multifocal–monosystemic Langherhans cell histiocytosis (LCH), formerly usually referred to as eosinophilic granuloma (EG) of bone. The condition developed in a 36-year-old man. A notable infrequent thoracic spine location and two successive distinct costal lesions were observed. Both the first costal site and the vertebral location healed spontaneously; the second costal lesion underwent biopsy resection. The patient’s disease course with an 8-year follow-up is discussed with reference to various treatment options, emphasising in selected cases a watchful conservative approach, in view of the widely documented potential for spontaneous healing. Received: 7 November 1997 / Accepted: 13 July 1998     

Oesophageal epithelial innervation in health and reflux oesophagitis

BACKGROUND: The response of the oesophagus to refluxed gastric contents is likely to depend on intact neural mechanisms in the oesophageal mucosa. The epithelial innervation has not been systematically evaluated in health or reflux disease. AIMS: To study oesophageal epithelial innervation in controls, and also inflamed and non-inflamed mucosa in patients with reflux oesophagitis and healed oesophagitis. PATIENTS: Ten controls, nine patients with reflux oesophagitis, and five patients with healed oesophagitis. METHODS: Oesophageal epithelial biopsy specimens were obtained at endoscopy. The distribution of the neuronal marker protein gene product 9.5 (PGP), and the neuropeptides calcitonin gene related peptide (CGRP), neuropeptide Y (NPY), substance P (SP), and vasoactive intestinal peptide (VIP) were investigated by immunohistochemistry. Density of innervation was assessed by the proportion of papillae in each oesophageal epithelial biopsy specimen containing immunoreactive fibres (found in the subepithelium and epithelial papillae, but not penetrating the epithelium). RESULTS: The proportion of papillae positive for PGP immunoreactive nerve fibres was significantly increased in inflamed tissue when compared with controls, and non-inflamed and healed tissue. There was also a significant increase in VIP immunoreactive fibres within epithelial papillae. Other neuropeptides showed no proportional changes in inflammation. CONCLUSIONS: Epithelial biopsy specimens can be used to assess innervation in the oesophagus. The innervation of the oesophageal mucosa is not altered in non-inflamed tissue of patients with oesophagitis but alters in response to inflammation, where there is a selective increase (about three- to fourfold) in VIP containing nerves.     

Eicosanoid synthesis in duodenal ulcer disease: decrease in leukotriene C4 by colloidal bismuth subcitrate.

The release of immunoreactive prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) from antral and duodenal mucosal biopsy specimens taken from 20 patients with duodenal ulcer disease was measured by radioimmunoassay before and four weeks after treatment with colloidal bismuth subcitrate. Gastroscopic and histological examination showed complete ulcer healing in 15/18 patients and duodenal histology looked normal (n = 15) or improved (n = 3): two patients failed to attend for a second endoscopy. Analysis of the supernatant from incubations of biopsy tissue in vitro showed that unstimulated antral release of PGE2 was significantly more than that from the duodenal mucosa (p less than 0.05), whereas basal release of LTC4 was significantly lower from antral biopsy specimens (p less than 0.05). Subsequent incubation of specimens with calcium ionophore A23187 caused an increase in LTC4 but not in PGE2 generation. The ability of antral and duodenal mucosa to form ionophore mediated LTC4 in patients with duodenal ulcer disease was significantly greater (p less than 0.05; p less than 0.01 respectively) than that of normal gastroduodenal mucosa. After colloidal bismuth subcitrate treatment, basal synthesis of PGE2 was unchanged in duodenal and antral specimens. In contrast, basal duodenal LTC4 was reduced (p less than 0.05), and the capacity for ionophore mediated duodenal LTC4 formation was substantially and significantly reduced after treatment (p less than 0.001). These results indicate that after therapeutic healing of duodenal ulcer (accompanied by clearance of inflammatory cell infiltrate), there is a reduced ability of duodenal mucosa to generate proinflammatory peptidoleukotrienes.     

Accumulation of apoptotic cells in the epidermis of patients with cutaneous lupus erythematosus after ultraviolet irradiation

OBJECTIVE: To examine whether apoptosis contributes to the pathogenesis of skin lesions in patients with cutaneous lupus erythematosus (CLE) after ultraviolet (UV) irradiation. METHODS: In situ nick translation and TUNEL were performed to detect apoptosis in 85 skin biopsy specimens from patients with various subtypes of CLE. Specimens from normal healthy donors and patients with polymorphous light eruption were used as controls. In addition to assessment of primary lesions, provocative phototesting was carried out to investigate events occurring secondary to UV irradiation during a very early stage of lesion formation. RESULTS: A significant increase in apoptotic nuclei was found in the upper epidermal layer of primary and UV light-induced skin lesions of CLE patients compared with controls. In tissue sections obtained from control subjects at 24 hours after a single exposure to UV light, a slight increase in the count of epidermal apoptotic nuclei was present as compared with skin tissue from CLE patients obtained under the same conditions before lesion formation. In sections obtained from controls at 72 hours after irradiation, a significant decrease in the apoptotic nuclei count was observed, consistent with a proper clearance of apoptotic cells in the period between 24 and 72 hours after irradiation. In striking contrast, the number of apoptotic nuclei increased significantly within this period in tissue sections from patients with CLE. CONCLUSION: These data support the hypothesis that apoptotic cells accumulate in the skin of patients with CLE after UV irradiation, as a result of impaired or delayed clearance. The nonengulfed cells may undergo secondary necrosis and release proinflammatory compounds and potential autoantigens, which may contribute to the inflammatory micromilieu that leads to formation of skin lesions in this disease.     

A case of primary esophageal tuberculosis diagnosed by identification of <Emphasis Type="Italic">Mycobacteria </Emphasis>in paraffin-embedded esophageal biopsy specimens by polymerase chain reaction

We present a case of primary esophageal tuberculosis diagnosed by polymerase chain reaction (PCR) of paraffin-embedded esophageal biopsy specimens. A 42-year-old Japanese woman visited our clinic because of dysphagia. Radiologic and endoscopic examinations revealed a stenotic lesion with reddish mucosa and multiple ulcers in the middle esophagus. There was no associated lesion outside the esophagus. Histological and bacteriological studies of esophageal biopsy specimens and gastric aspirates did not give a definitive diagnosis. However, mycobacterial DNA was detected by PCR of paraffin-embedded esophageal biopsy specimens. She then was diagnosed as having primary esophageal tuberculosis. The esophageal mucosal lesion almost healed after 1 month of antituberculosis medication with residual annular stenosis which was resolved later by endoscopic balloon dilation. Received: May 25, 2001 / Accepted: December 14, 2001 Acknowledgments. The authors thank Chiaki Sato (Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, Tochigi, Japan) for technical support. Reprint requests to: T. Fujiwara     

Tumour necrosis factor alpha is expressed in refractory skin lesions from patients with subacute cutaneous lupus erythematosus

OBJECTIVES: To investigate whether tumour necrosis factor alpha (TNFalpha) is expressed in subacute cutaneous lupus erythematosus (SCLE) skin lesions. METHODS: The in situ expression of TNFalpha in refractory lesional and non-lesional skin biopsy specimens from patients with SCLE was analysed using an immunohistochemical approach. At the time of biopsy these patients were receiving treatment with systemic medications such as antimalarial agents, immunosuppressive drugs, and thalidomide. Expression of TNFalpha was also evaluated in cutaneous lesions of patients with other inflammatory and neoplastic skin diseases as controls. RESULTS: The data showed that refractory lesional skin tissue from patients with SCLE displays a strongly positive distribution of TNFalpha, particularly within the epidermis. No prominent staining was seen in non-lesional skin from the same group of patients or in cutaneous lesions from the control group. CONCLUSIONS: These findings suggest that TNFalpha is localised and produced by epidermal cells within SCLE skin lesions and support its potential role in the pathogenesis of SCLE. The tissue localisation of TNFalpha may represent a potential therapeutic target providing a new perspective in the treatment of refractory skin lesions in patients with SCLE.     

Monochloramine impairs mucosal blood flow response and healing of gastric lesions in rats: relation to capsaicin-sensitive sensory neurons

AIMS:We examined the effects of monochloramine (NH2Cl) on the gastric mucosal blood flow (GMBF) response and the healing of ethanol-induced gastric lesions in rats. METHODS: Rats fasted for 18 h were given the 99% ethanol p.o. for induction of gastric lesions, and were fed normally from 1 h later onwards. Monochloramine, at non-ulcerogenic doses (5 to approximately 20 mmol/L), was given p.o. twice daily for 7 days, starting 2 h after ethanol treatment. RESULTS: Gastric lesions caused by ethanol healed almost completely within 7 days with re-epithelialization. The repeated administration of NH2Cl significantly delayed the healing of ethanol-induced gastric lesions in a dose-dependent manner. The damaged mucosa showed a marked rise in H+ permeability, resulting in luminal acid loss, but this process was accompanied by an increase of mucosal blood flow. Monochloramine did not affect the increased mucosal H+ permeability observed in the stomach after damage by ethanol, but significantly inhibited the mucosal hyperemic response associated with luminal acid loss. Prior exposure of the mucosa to NH2Cl (20 mmol/L) did not affect the gastric hyperemic response caused by mucosal application of misoprostol (a prostaglandin E1 derivative) or NOR-3 (a nitric oxide donor), but totally attenuated the increase of GMBF in response to intragastric capsaicin. Impaired healing and GMBF responses were also observed in rats following chemical ablation of capsaicin-sensitive sensory neurons. CONCLUSIONS: These results suggest that NH2Cl impaired the healing of acute gastric mucosal lesions at low concentrations, and this action may be attributable, at least partly, to the impairment of gastric hyperemic response caused by the dysfunction of capsaicin-sensitive sensory neurons.     

The effects of stress on wound healing in male tree lizards (Urosaurus ornatus)

Exposure to stress can affect an organism's partitioning of resources among immune function and other organismal functions. However, measuring immune function is often difficult. Recent studies show that the rate of cutaneous wound healing in laboratory rodents is a simple, integrated measure of stress-sensitive immune function. We investigated the use of this technique in tree lizards to test the hypotheses (1) that stress compromises wound healing and (2) that this effect is at least partially mediated by corticosterone. Laboratory-housed male tree lizards randomly assigned to the experimental and control treatment groups received a 3.5 mm cutaneous biopsy on the dorsal surface of the pelvis. Experimental group males were restrained in cloth bags for 60 min every day for 21 days during the healing profile, whereas control males were left in their cages. Wound sizes were measured every other day by image analysis. Control animals healed faster than stressed animals. The difference in wound surface area between the groups was most pronounced early in the healing profile. Stressed animals also had higher corticosterone levels and corticosterone was negatively correlated with healing rate in the stressed animals. These observations support both hypotheses that stress compromises healing and that corticosterone may act to mediate the effects of stress.     

Pathology resulting from the administration of a live attenuated anti-Schistosoma haematobium vaccine in baboons

Baboons (Papio anubis) were injected in the leg muscle with 18,000 20 Krad irradiated schistosomula of Schistosoma haematobium. Four protocols were followed: single, primary injection; single injection into animals primed by patent S. haematobium infection; secondary vaccine injection following an earlier injection; and single injection following praziquantel treatment of infected animals. Injection of the putative vaccine elicited localized mixed inflammatory infiltration at the site of injection which was both intense and prolonged. Three grades of tissue reaction were seen: the relatively mild primary response; the response in infected animals which had enhanced tissue eosinophilia; and the response in animals primed by prior injection and drug-treated prior infection. The latter 2 showed intensification of eosinophilia, stellate abscesses in the lesion centers, and perischistosomular Hoeppli precipitates. Intramuscular lesions peaked at 14 days for the primary response and at 7 days for all secondary responses. Traces of the milder lesions persisted beyond 4 weeks; more severe reactions healed more rapidly. Some schistosomula survived for 14 days in the milder reactions. A few larvae were deposited in the skin by backflushing of the injectate which produced local inflammation. Compared to mice, live schistosome vaccines injected into baboons elicited greater local inflammation; however, while evidence suggested that sporadic vaccine schistosomula did reach the lymphatic nodes draining the injection sites, no systemic lesions were found and the injection sites healed in approximately 5-6 weeks without permanent damage.