In vitro assessment of nanomedicines' propensity to cause palmar-plantar erythrodysesthesia: A Doxil vs. doxorubicin case study.

Palmar-plantar erythrodysesthesia (PPE), also known as hand and foot syndrome, is a condition characterized by inflammation-mediated damage to the skin on the palms and soles of the hands and feet. PPE limits the successful therapeutic applications of anticancer drugs. However, identifying this toxicity during preclinical studies is challenging due to the lack of accurate in vitro and in vivo animal-based models. Therefore, there is a need for reliable models that would allow the detection of this toxicity early during the drug development process. Herein, we describe the use of an in vitro skin explant assay to assess traditional DXR, Doxil reference listed drug (RLD) and two generic PEGylated liposomal DXR formulations for their abilities to cause inflammation and skin damage. We demonstrate that the results obtained with the in vitro skin explant assay model for traditional DXR and Doxil correlate with the clinical data.

Mycobacterial antigens accumulation in foamy macrophages in murine pulmonary tuberculosis lesions: Association with necrosis and making of cavities.

Understanding mechanisms of cavitation in tuberculosis (TB) is the missing link that could advance the field towards better control of the infection. Descriptions of human TB suggest that postprimary TB begins as lipid pneumonia of foamy macrophages that undergoes caseating necrosis and fragmentation to produce cavities. This study aimed to investigate the various mycobacterial antigens accumulating in foamy macrophages and their relation to tissue destruction and necrosis. Pulmonary tissues from mice with slowly progressive TB were studied for histopathology, acid-fast bacilli (AFB) and presence of mycobacterial antigens. Digital quantification using Aperio ImageScope was done. Until week 12 postinfection, mice were healthy, and lesions were small with scarce AFB and mycobacterial antigens. Colony-forming units (CFUs) increased exponentially. At week 16-33, mice were sick, macrophages attained foamy appearance with an increase in antigens (P < .05), 1.5 log increase in CFUs and an approximately onefold increase in AFB. At week 37-41, mice started dying with a shift in morphology towards necrosis. A >20-fold increase in mycobacterial antigens was observed with only less than one log increase in CFUs and sevenfold increase in AFB. Secreted antigens were significantly (P < .05) higher compared to cell-wall antigens throughout infection. Focal areas of necrosis were associated with an approximately 40-fold increase in antigen MPT46, functionally active thioredoxin, and a significant increase in all secreted antigens. In conclusion, mycobacterial antigens accumulate in the foamy macrophages in TB lesions during slowly progressive murine pulmonary TB. Secreted antigens and MPT46 correlated with necrosis, thereby implying that they might trigger the formation of cavities.

Diagnosis of extrapulmonary tuberculosis using the MPT64 antigen detection test in a high-income low tuberculosis prevalence setting.

BACKGROUND: Extrapulmonary tuberculosis (EPTB) poses diagnostic challenges due to the paucibacillary nature of the disease. The immunochemistry-based MPT64 antigen detection test (MPT64 test) has shown promising results for diagnosing EPTB in previous studies performed in low-resource settings, with higher sensitivity than microscopy and culture. The aim of this study was to investigate the performance of the MPT64 test in a routine clinical setting in a high-income low TB prevalence country. METHODS: Extrapulmonary samples sent for TB diagnostics to microbiology and pathology laboratories at three regional tertiary care hospitals in Norway in a one-year period were included and subjected to the MPT64 test in parallel to the routine TB diagnostic tests. RESULTS: Samples from 288 patients were included and categorised as confirmed TB cases (n = 26), clinically diagnosed TB cases (n = 5), non-TB cases (n = 243) and uncategorised (n = 14), using a composite reference standard (CRS). In formalin-fixed biopsies, the sensitivity (95% CI) of the MPT64 test, microscopy, PCR-based tests pooled, and culture was 37% (16-62), 20% (4-48), 37% (16-62) and 50% (23-77), respectively, against the CRS. The MPT64 test showed a good positive predictive value (88%) and an excellent specificity (99, 95% CI 92-100) in formalin-fixed biopsies. In fine-needle aspirates, pus and fluid samples, the test performance was lower. CONCLUSIONS: The MPT64 test was implementable in pathology laboratories as part of routine diagnostics, and although the sensitivity of the MPT64 test was not better than culture in this setting, the test supplements other rapid diagnostic methods, including microscopy and PCR-based tests, and can contribute to strengthen the diagnosis of EPTB in formalin-fixed biopsies in the absence of culture confirmation.

Evaluation of treatment response in extrapulmonary tuberculosis in a low-resource setting.

BACKGROUND: Diagnosing extrapulmonary tuberculosis (EPTB) is challenging and many patients are initiated on empirical anti-TB treatment without a laboratory confirmed diagnosis. Monitoring treatment response is thus important to ensure correct diagnosis and proper disease management. The definition of satisfactory response to treatment in EPTB remains unclear. The objectives of this study were to describe the clinical presentation of EPTB and the effect of treatment on clinical parameters. Further, to assess if simple clinical parameters, without laboratory data, could evaluate treatment response. METHODS: Prospective cohort study of presumptive EPTB patients at Mnazi Mmoja Hospital, Zanzibar. By using a composite reference standard, patients were categorized as TB or non-TB cases. The TB patients were followed during anti-TB treatment. RESULTS: There were 64 TB and 62 non-TB cases. The frequency of symptoms at baseline were comparable in TB and non-TB patients, with lymphadenitis and pleuritis as the most common manifestations. Among TB cases, there was a trend towards regression of lymphadenopathy after 2 months, and at treatment completion 24/28 (86%) cases showed full regression. Weight gain ≥5% was reported in 36/49 (73%) of the TB patients at 2 months and in 38/46 (83%) at treatment completion. After 2 months of treatment, a combination of clinical parameters; improvement of symptoms (50/50), ≥5% weight gain (36/49) and regression of physical signs (45/49) correlated with the treatment response. CONCLUSIONS: An algorithm including only simple clinical parameters could be used as an easy tool to assess treatment responses in low-resource settings. However, this needs to be tested on a larger sample size.

Epidemiological and histopathological profile of malignant melanoma in Malawi.

BACKGROUND: Studies on malignant melanoma have largely focused on Caucasian populations due to higher incidence in lighter-skinned individuals. While there is a well developed body of literature describing melanoma in African-Americans, much less is known about melanoma in black Africans. Prior reports have suggested that it is reportedly extremely rare in black Africans who are considered to mostly have the acral lentiginous subtype. However, an accurate understanding of melanoma in this part of the world is hindered by the very limited nature of prior publications. The aim of this study was to determine the epidemiological profile, anatomical distribution and histopathological features of melanoma presenting in Africans at a tertiary referral hospital in Malawi. METHODS: This is a retrospective study that characterized melanoma cases diagnosed from January 2012 to December 2017, at a cancer referral centre in Malawi. All confirmed, malignant melanoma cases during the study period were retrieved. Data abstracted included age, sex, anatomic site and whether it was a primary or metastatic site. Breslow thickness in millimetres, Clark level of invasion, presence of ulceration and melanoma subtype were also evaluated. RESULTS: One hundred thirty-two cases were included in the study, 81 (61%) were female and 26 (20%) were from a metastatic site. The mean age was 57 years (sd = 15) with the majority in the age group 60-69 years. Males presented at an older age than females. Ninety five percent of cutaneous melanomas were located on acral sites, most commonly the foot (87%) and the most common histopathological subtype was acral lentiginous. Eighty four percent presented with a Breslow thickness over 4 mm (median 9 mm). CONCLUSION: Our study shows that malignant melanoma occurs in black people in Malawi and may be an under-appreciated malignancy. While long term clinical follow-up was not available, most patients presented at late stages of the disease, supporting a poor prognosis. These results suggest that increased awareness of melanoma in black Africans and earlier intervention may have meaningful impacts on outcomes and survival.

Differential in vivo expression of mycobacterial antigens in Mycobacterium tuberculosis infected lungs and lymph node tissues.

BACKGROUND: The clinical course of tuberculosis (TB) infection, bacterial load and the morphology of lesions vary between pulmonary and extrapulmonary TB. Antigens expressed in abundance during infection could represent relevant antigens in the development of diagnostic tools, but little is known about the in vivo expression of various M. tuberculosis antigens in different clinical manifestations. The aim of this study was to study the differences in the presence of major secreted as well as somatic mycobacterial antigens in host tissues during advanced rapidly progressing and fatal pulmonary disease with mainly pneumonic infiltrates and high bacterial load, and to compare this to the presence of the same antigens in TB lymphadenitis cases, which is mainly chronic and self-limiting disease with organised granulomas and lower bacterial load. METHODS: Human pulmonary (n = 3) and lymph node (n = 17) TB biopsies, and non-TB controls (n = 12) were studied. Ziehl-Neelsen stain, nested PCR 1S6110 and immunohistochemistry were performed. Major secreted (MPT32, MPT44, MPT46, MPT51, MPT53, MPT59, MPT63, and MPT64) and somatic mycobacterial antigens (Mce1A, Hsp65, and MPT57) were detected by using rabbit polyclonal antibodies. RESULTS: Plenty of bacilli were detectable with Ziehl-Neelsen stain in the lung biopsies while no bacilli were detected in the lymph node biopsies. All the cases were shown to be positive by PCR. Both secretory and somatic antigens were expressed in abundance in pulmonary infiltrates, while primarily somatic antigens were detected in the lymphadenitis cases. Of the secreted antigens, only MPT64 was consistently detected in both cases, indicating a preferential accumulation of this antigen within the inflammatory cells, even if the cells of the granuloma can efficiently restrict bacterial growth and clear away the secreted antigens. CONCLUSIONS: This study shows that major secreted mycobacterial antigens were found in high amounts in advanced pulmonary lesions without proper granuloma formation, while their level of staining was very low, or absent, in the lymph node TB lesions with organised granulomas and very low bacillary load, with one exception of MPT64, suggesting its role in the persistence of chronic infection. These findings have implication for development of new diagnostic tools.

Immunocytochemical detection of Mycobacterium Tuberculosis complex specific antigen, MPT64, improves diagnosis of tuberculous lymphadenitis and tuberculous pleuritis.

BACKGROUND: A rapid, sensitive and accurate laboratory diagnosis is of prime importance in suspected extrapulmonary tuberculosis (EPTB) cases. However, traditional techniques for the detection of acid-fast bacilli have limitations. The aim of the study was to evaluate the diagnostic value of immunocytochemical staining for detection of Mycobacterium tuberculosis complex specific antigen, MPT64, in aspirates from pleural effusions and lymph nodes, the most common presentations of EPTB. METHOD: A cross-sectional study was conducted by including patients at Tikur Anbessa Specialized Hospital and the United Vision Medical Services from December 2011 to June 2012. Lymph node aspirates and pleural fluid samples were collected and analyzed from a total of 51 cases (26 tuberculous (TB) pleuritis and 25 TB lymphadenitis) and 67 non-TB controls. Each specimen was subjected to Ziehl-Neelsen (ZN) staining, culture on Lowenstein- Jensen (LJ) medium, cytological examination, Polymerase Chain Reaction (PCR) using IS1081gene sequence as a primer and immunocytochemistry (ICC) with polyclonal anti-MPT64 antibody. All patients were screened for HIV. RESULT: ICC was positive in 38 of 51 cases and in the 7 of 67 controls giving an overall sensitivity and specificity of 74.5% and 89.5%, respectively. Using IS1081-PCR as a reference method, the sensitivity and specificity, positive and negative predictive value of ICC was 88.1%, 89.5%, 82.2% and 93.2%, respectively. The case detection rate increased from 13.7% by ZN stain to 19.6% by LJ culture, to 66.7% by cytology and 74.5% by ICC. CONCLUSION: Immunocytochemistry with anti-MPT64 antigen improved detection of TB in pleural effusion and lymph node aspirates. Further studies using monoclonal antibodies on samples from other sites of EPTB is recommended to validate this relatively simple diagnostic method for EPTB.

CD11c(+) dendritic cells rather than Langerhans cells are reduced in normal skin of immunosuppressed renal transplant recipients.

The increased risk of squamous cell carcinomas (SCC) in renal transplant recipients (RTR) is related to impaired immunosurveillance as a consequence of immunosuppressive therapy. Since dendritic cells (DC) play an important role in immunosurveillance, we investigated the quantity of DC subsets and macrophages in normal skin of RTR and immunocompetent controls by immunohistochemistry. In this comparative study Langerhans' cells (LC) were present in similar numbers in RTR and controls. The number of CD11c+ DC was significantly reduced in RTR, particularly in patients on triple treatment therapy, compared with controls. Macrophages were significantly increased. Plasmacytoid DC were not detected in normal skin. The reduced quantity of CD11c+ DC and increased number of macrophages in normal skin of immunosuppressed RTR may contribute to the increased incidence of SCC in RTR. This finding underlines the role of DC subsets in immunosurveillance, and may have implications for our understanding of the effect of immunosuppression on DC subsets.

Cause of death in patients with tuberculosis: A study based on epidemiological and autopsy records of Western Norway 1931-47.

BACKGROUND: Without treatment, nearly 50 % of tuberculosis (TB) patients die. World Health Organization's definition of TB deaths does not take into consideration whether the cause of death was TB or other non-TB co-morbid conditions. We aimed to improve our knowledge of the causes of death in patients with TB. METHODS: Single-center retrospective study conducted at Gade Institute of Pathology, Haukeland University Hospital, Bergen, Norway. Autopsy data of 269 patients with TB was collected from autopsy journals, and epidemiological data was collected from Norwegian Official Statistics books for period 1931-1947. RESULTS: Of all TB deaths reported in epidemiological reports, pulmonary TB accounted for 81 % and extrapulmonary TB for 19 %. However, in autopsy records, only 21 % of cases with active pulmonary TB died because of TB. Extrapulmonary involvement was significantly associated with higher mortality (OR EPTB as compared to PTB; 3.27, CI 1.91 - 5.61) and constituted 79 % of deaths attributable to TB. A significant burden of extrapulmonary TB was found in autopsy records (63 %), while in epidemiological records, only 4 % of cases were reported. CONCLUSIONS: Extrapulmonary involvement was a predictor of mortality due to TB in hospitalized TB patients. The contribution of extrapulmonary TB to TB mortality seems to be underestimated because extrapulmonary TB largely remains underdiagnosed and underreported in epidemiological data.

Novel Insights into the Pathogenesis of Human Post-Primary Tuberculosis from Archival Material of the Pre-Antibiotic Era, 1931-1947.

OBJECTIVES: Primary and post-primary tuberculosis (TB) are distinct entities. The aim of this study was to study the histopathology of primary and post-primary TB by using the unique human autopsy material from the pre-antibiotic era, 1931-1947. MATERIAL AND METHODS: Autopsy data were collected from the autopsy journals, and the human tissue was collected from the pathology archives at the Department of Pathology, the Gades Institute. RESULTS: Histological presentations of TB lesions showed great diversity within a single lung. Post-primary TB starts as a pneumonia forming early lesions, characterized by the infiltration of foamy macrophages containing mycobacterial antigens within alveoli, and progressing to necrotic pneumonias with an increasing density of mycobacterial antigens in the lesions. These necrotic pneumonic lesions appeared to either resolve as fibrocaseous lesions or lead to cavitation. The typical granulomatous inflammation, the hallmark of TB lesions, appeared later in the post-primary TB and surrounded the pneumonic lesions. These post-primary granulomas contained lesser mycobacterial antigens as compared to necrotic pneumonia. CONCLUSIONS: Immunopathogenesis of post-primary TB is different from primary TB and starts as pneumonia. The early lesions of post-primary TB may progress or regress, holding the key to understanding how a host can develop the disease despite an effective TB immunity.

Improved diagnosis of extrapulmonary tuberculosis in adults with and without HIV in Mbeya, Tanzania using the MPT64 antigen detection test.

Extrapulmonary tuberculosis (EPTB) in People Living with HIV (PLWHIV) is a diagnostic challenge. Our immunochemistry based MPT64 antigen detection test has shown improved sensitivity compared to current laboratory tests in the resource limited diagnostic setting. The aim of this study was to validate the implementability and diagnostic performance of the test in PLWHIV and HIV negative adults in a HIV endemic Tanzanian setting. Adult (>18 y) presumptive EPTB patients were prospectively enrolled at Mbeya Zonal Referral Hospital and followed to the end of treatment or until an alternative diagnosis was reached. Suspected sites of infection were sampled and were subject to routine diagnostics, GeneXpert MTB/RIF assay and the MPT64 test. The performance of the diagnostics tests was assessed using a composite reference standard that included clinical suspicion, mycobacterial culture, response to anti-tuberculosis (TB) therapy, cytological and radiological findings. Patients (N = 168) were categorized as 21 confirmed TB, 23 probable TB and 44 possible TB cases, 69 patients were categorized as non-TB cases and 11 were uncategorized. In the TB group, the three most common infections were adenitis (41%), peritonitis (19%) and pleuritis (14%). The TB and non-TB groups did not differ in HIV seropositivity (46% vs 42%) Among HIV negative and PLWHIV, the MPT64 test had a sensitivity of (91% vs 78%), specificity (75% vs 86%), positive predictive value (80% vs 88%), negative predictive value (89% vs 74%), and accuracy (84% vs 81%), respectively. Performance was not significantly reduced in PLWHIV, and sensitivity was higher than in the currently used tests, including the GeneXpert MTB/RIF assay. The MPT64 test improved the diagnosis of EPTB, irrespective of HIV status. The test performed better than currently used diagnostic test. The test was implementable in a tertiary level hospital with basic pathology services in a HIV endemic Tanzanian setting.

The value of histological examination in the diagnosis of tuberculous lymphadenitis in the era of rapid molecular diagnosis.

Extrapulmonary tuberculosis often poses a diagnostic challenge. This study aimed to assess the value of histological examination in diagnosing tuberculous lymphadenitis (LNTB) when performed simultaneously with rapid molecular assay (Xpert MTB/RIF) testing. People presumed to have LNTB were prospectively enrolled in a tertiary care hospital. Excision biopsy was performed and tested by histology, Xpert, and culture. Of 390 lymph nodes, 11 (2.8%) were positive by AFB microscopy, 124 (31.8%) by Xpert, 137 (35.1%) by culture, and histopathology was consistent with TB in 208 (53.3%). Altogether, LNTB was diagnosed in 228 and bacteriologically confirmed TB in 178 cases. Against culture, histopathology versus Xpert had higher sensitivity (93 vs. 62%) but lower specificity (68 vs. 83%). In patients with short clinical history, a significantly higher number of Xpert-positive specimens were culture-positive. Among patients with histology suggestive of TB, no difference was seen in response to treatment between bacteriology positive and negative, but a significant slow response was noted in bacteriology confirmed TB with nonspecific histology. In a country like Pakistan, with high TB and low HIV prevalence, diagnosis is possible for more than 95% of LNTB when Xpert and histopathology examination is used in combination, compared to less than 60% by Xpert alone.

In situ dissection of the graft-versus-host activities of cytotoxic T cells specific for minor histocompatibility antigens.

Minor histocompatibility antigens (mHags) are immunogenic peptides from polymorphic cellular proteins that induce strong T-cell responses after human leukocyte antigen (HLA)-matched, mHag-mismatched stem-cell transplantation. mHags with broad or limited tissue expression are target antigens for graft-versus-host (GvH) and graft-versus-leukemia (GvL) reactivities. Separation of these activities is crucial for adoptive immunotherapy of leukemia without GvH disease. Therefore, using a skin-explant assay we investigated the in situ activities of cytotoxic T lymphocytes (CTLs) specific for the ubiquitously expressed mHag H-Y and for the hematopoietic-restricted mHags HA-1 and HA-2. H-Y-specific CTLs, visualized by tetrameric HLA-mHag peptide complexes, infiltrated male skin sections within 24 hours, induced severe GvH reactions of grade III-IV and produced high levels of IFN-gamma. In contrast, CTLs specific for the hematopoietic system-specific mHags HA-1 and HA-2 induced no or low GvH reactions above background and produced little or no interferon-gamma, unless the skin sections were preincubated with HA-1/HA-2 synthetic peptides. These results provide the first in situ dissection of GvH effects by mHag-specific CTLs and show that ubiquitously expressed mHags are the prime targets of GvH disease.

Immunochemistry-Based Diagnosis of Extrapulmonary Tuberculosis: A Strategy for Large-Scale Production of MPT64-Antibodies for Use in the MPT64 Antigen Detection Test.

Tuberculosis (TB) is a global health problem. The immunohistochemistry (IHC)-based MPT64 antigen detection test has shown promising results for diagnosing extrapulmonary TB in previous studies. However, the anti-MPT64 antibody currently used in the test is in limited supply, and reproduction of a functional antibody is a prerequisite for further large-scale use. Various antigen-adjuvant combinations and immunisation protocols were tested in mice and rabbits to generate monoclonal and polyclonal antibodies. Antibodies were screened in IHC, and the final new antibody was validated on clinical human specimens. We were not able to generate monoclonal antibodies that were functional in IHC, but we obtained multiple functional polyclonal antibodies through careful selection of antigen-adjuvant and comprehensive screening in IHC of both pre-immune sera and antisera. To overcome the limitation of batch-to-batch variability with polyclonal antibodies, the best performing individual polyclonal antibodies were pooled to one final large-volume new anti-MPT64 antibody. The sensitivity of the new antibody was in the same range as the reference antibody, while the specificity was somewhat reduced. Our results suggest that it possible to reproduce a large-volume functional polyclonal antibody with stable performance, thereby securing stable supplies and reproducibility of the MPT64 test, albeit further validation remains to be done.

MPT64 antigen detection test improves diagnosis of pediatric extrapulmonary tuberculosis in Mbeya, Tanzania.

Pediatric extrapulmonary tuberculosis (EPTB) is a diagnostic challenge. A new immunochemistry based MPT64 antigen detection test has shown improved sensitivity compared to current laboratory tests. The aim of this study was to implement and validate the test performance in a resource limited African setting. Presumptive pediatric (0-18 y) EPTB patients were prospectively enrolled at Mbeya Zonal Referral Hospital, and followed to the end of treatment or until a final diagnosis was reached. Specimens from suspected sites of infection were subject to routine diagnostics, GeneXpert MTB/RIF assay and the MPT64 test. The performance of the tests was assessed using mycobacterial culture as well as a composite reference standard. 30 patients were categorized as TB cases, 31 as non-TB cases and 2 were uncategorized. In the TB group, the three most common infections were adenitis (30%), peritonitis (30%) and meningitis (20%). The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the MPT64 test was 92%, 88%, 87%, 92% and 90%, respectively. Mortality was equally high among TB/non-TB cases (23% vs 21%), and malnutrition was the main comorbidity among TB cases. The MPT64 test was implementable in the routine diagnostics in a low-resource setting and improved the diagnosis of pediatric EPTB.

[Hidradenitis suppurativa]. / Hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa is a chronic inflammatory skin disease characterized by recurrent tender nodules and boils, usually in the armpits and groins. Draining fistulas and hypertrophic scarring are hallmarks of more severe disease. The objective of this article is to review the clinical presentation, diagnostic considerations and treatment of the disease. MATERIAL AND METHODS: The article is based on a non-systematic literature search in PubMed, review of dermatology textbooks and the author's personal clinical experience. RESULTS: Hidradenitis suppurativa, also known as acne inversa, is a follicular occlusion disease that can severely reduce quality of life. Staphylococci and other pathogenic bacteria frequently colonize the lesions, but the disease is not primarily a bacterial infection. Smoking and obesity can worsen disease activity. Moderate and severe disease is usually treated with excisional surgery. Antibiotics, often tetracyclines, are indicated for mild disease and as an adjunct to surgery in more severe disease. Antibiotics, however, are not curative. New treatment options, such as TNF-alpha inhibitors and zinc gluconate should still be considered experimental. INTERPRETATION: Hidradenitis suppurativa is probably underdiagnosed. The disease is often recalcitrant to treatment. The effect of medical treatment is not supported by high quality evidence.

[Nephrogenic systemic fibrosis and use of MR contrast media]. / Nefrogen systemisk fibrose ved bruk av MR-kontrastmiddel.

BACKGROUND: Nephrogenic systemic fibrosis (NSF) is a new and probably iatrogenic disorder. It was first described time in 2000 and associated with the use of gadolinium containing MR contrast media in patients with impaired renal function in 2006. MATERIAL AND METHOD: We describe a case of NSF identified at Haukeland University Hospital and give a brief overview of the disease, based on literature retrieved from a non-systematic search. RESULTS AND INTERPRETATION: NSF is a serious systemic disorder with active inflammation and fibrosis, particularly in skin, but also in other tissue such as skeletal muscle, heart and oesophagus. The condition is very difficult to treat. Awareness of the illness has led to changes in guidelines and clinical practice regarding use of gadolinium-containing contrast media in patients with renal impairment.

Correlation of Hsp70-1 and Hsp70-2 gene expression with the degree of graft-versus-host reaction in a rat skin explant model.

BACKGROUND: Graft-versus-host disease (GVHD) is the most serious complication after allogeneic hematopoietic stem-cell transplantation. A human skin explant assay has been used to predict the risk of GVHD in patients by histological grading of graft-versus-host reactions (GVHR). New molecular markers of GVHR might help to further increase the predictive value of the assay. METHODS: A rat skin explant assay has been developed to further aid in identifying potential novel molecular markers. RESULTS: In inbred rat strains GVHR were observed in skin explants co-cultured with allogeneic lymphocytes stimulated against minor or major histocompatibility antigens. The histological signs of GVHR were similar to those observed in human skin explant assays and acute GVHD lesions occurring in rats after experimental bone marrow transplantation. Heat shock protein (HSP) 70 has been shown to be expressed during GVHR. We therefore investigated the expression of the three major histocompatibility complex (MHC)-linked HSP70 genes in rat skin explants. The two major stress-inducible genes Hsp70-1 and Hsp70-2 were found to be upregulated in the allogeneic rat skin explant assays. The increase in mRNA correlated with the GVHR grade (I-IV). Interestingly, the expression of the third MHC-linked Hsp70 gene Hsp70-3 was not found to be augmented during GVHR. CONCLUSION: The observed induction of the MHC-encoded Hsp70-1 and Hsp70-2 genes might serve as new markers of GVHR and as potentially novel diagnostic tools for GVHD.

Differential expression of mycobacterial antigen MPT64, apoptosis and inflammatory markers in multinucleated giant cells and epithelioid cells in granulomas caused by Mycobacterium tuberculosis.

The development of granulomas is a major histopathological feature of tuberculosis. Very little information is available concerning the physiology and functions of different cell types in the tuberculous granulomas. The aim of this study was to compare the epithelioid cells (ECs) and multinucleated giant cells (MGCs) in the granulomas caused by Mycobacterium tuberculosis complex organisms. Lymph node biopsies from 30 cases of lymphadenitis were studied for expression of the secreted mycobacterial protein MPT64, caspase 3 as a marker of apoptosis, apoptosis-related proteins (Fas Ligand, Fas and Bax) and inflammatory cytokines (interleukin-10, transforming growth factor-beta (TGF-beta), tumour necrosis factor-alpha and interferon-gamma) by immunohistochemistry. MGCs more often contained M. tuberculosis secretory antigen MPT64 (p < 0.001) and expressed more TGF-beta (p = 0.004) than ECs. The total number of apoptotic MGCs was higher than the number of apoptotic ECs (p = 0.04). Interestingly, there was a significant negative correlation between apoptosis and MPT64 expression in MGCs (r = -0.569, p = 0.003), but not in ECs, implying that the heavy antigen load would lead to inhibition of apoptosis in these cells. When compared with ECs, higher percentage of MGCs expressed Fas Ligand and Fas (p < 0.004). The role of MGCs may thus be different from surrounding ECs and these cells by virtue of higher mycobacterial antigen load, more TGF-beta and reduced apoptosis may contribute towards persistence of infection.

Diagnostic delay in extrapulmonary tuberculosis and impact on patient morbidity: A study from Zanzibar.

BACKGROUND: Early and proper treatment of tuberculosis could have an important impact on the morbidity, mortality and the economic situation of patients. There is insufficient knowledge on the extent of diagnostic delay and the associated factors in extrapulmonary tuberculosis (EPTB). The aims of this study were to assess the health care seeking behaviour, EPTB knowledge and diagnostic delay in presumptive EPTB patients at the main referral hospital in Zanzibar, factors associated with longer delay, and the impact of untreated EPTB on self-rated health. MATERIALS AND METHODS: Prospective data collection using a semi-structured questionnaire in patients presenting with symptoms suggestive of EPTB. The time between the onset of symptoms and first visit to a health care provider (patient delay), and then to the initiation of treatment (health system delay) and total delay were analysed according to sociodemographic and clinical factors and health care seeking trajectories. The EQ-5D-3L was used among the adult EPTB patients to assess the impact of treatment on self-rated health. RESULTS: Of the 132 patients with median age of 27 years (interquartile range 8-41), 69 were categorized as TB cases and 63 as non-TB cases. The median patient, health system and total delays were 14, 34 and 62 days respectively, among the EPTB patients. A longer health system delay with repeated visits to the same health care level was reported. Significantly better self-rated health status was described after treatment. The knowledge regarding extrapulmonary disease was low. CONCLUSION: Many EPTB patients, presenting to the main referral hospital in Zanzibar, experience a long delay in the initiation of treatment, specially patients with TB lymphadenitis. The health system delay is the major contributor to the total delay. The improvement of self-rated health after treatment implies that timely treatment has the potential to reduce morbidity and the economic loss for the patient.