Role of fibrogenic markers in chronic hepatitis C and associated hepatocellular carcinoma.

Detection and follow up of fibrogenesis in chronic hepatitis C (CHC) is mandatory for early treatment and risk stratification. The current study included 120 patients with CHC, of whom 30 had liver cirrhosis (LC) and 30 had hepatocellular carcinoma (HCC). 15 wedge liver biopsies, taken during laparoscopic cholecystectomy, were included as normal controls. Cases were subjected to laboratory investigations, serologic markers for viral hepatitis and assessment of circulating levels of hyaluronic acid (HA) and platelet-derived growth factor (PDGF). Immunohistochemical expression of connective tissue growth factor (CTGF), PDGF and transforming growth factor-ß1 (TGF-ß1) was also carried out. A significant increase (p < 0.01) in serum HA was noticed in CHC, LC and HCC compared to controls. Although, a significant decrease in serum PDGF was detected in CHC and LC compared to controls, HCC values were comparable. A significant up-regulation of CTGF was detected in CHC, LC and HCC (p < 0.01) in contrast to its limited mild expression in normal livers. Intense PDGF positive staining was noticed in CHC, LC and HCC compared to scattered faint expression in controls. The significant expression and marked intensity of PDGF staining matched the progress to tumorigenesis. A positive TGF-ß1 immunostaining was also noticed in CHC, LC and HCC. An intense and extensive cytoplasmic expression of TGF-ß1 was encountered in patients with LC revealing that CTGF, PDGF and TGF-ß1 act synergistically in LC. Data revealed that HA and CTGF may be implicated as important diagnostic parameters for assessment of hepatic fibrosis and PDGF for monitoring malignant transformation in CHC.

Electron microscopy study of peripheral nerves in the uvulae of snorers and obstructive sleep apnoea patients.

HYPOTHESIS: The pathophysiology of snoring and obstructive sleep apnoea is still unclear. Two theories are proposed. The first is the obstructive theory, which postulates palatopharyngeal muscle hypertrophy leading to airway narrowing; there is no neural role. The second is the neurogenic theory, which postulates neural degeneration due to vibratory stretch trauma, leading to muscle atrophy and collapse. As identification of nerve fibres in the uvula and palate is difficult and time-consuming, all previous studies aiming to differentiate between these two theories have been based on indirect observation of the muscles, rather than direct study of the nerves. METHODS: We conducted a prospective study to directly observe and study nerve fibres in uvular specimens from 10 cases of obstructive sleep apnoea, compared with specimens from 10 cases of simple snoring, using transmission electron microscopy. Five autopsy cases served as controls. RESULTS: Obstructive sleep apnoea was associated with definite degenerative changes in myelinated and unmyelinated nerve endings. These degenerative changes were present to a lesser degree and in a smaller proportion of cases of simple snoring. CONCLUSION: The events postulated by the neurogenic theory of obstructive sleep apnoea appear to play an important role in the pathophysiology of snoring and obstructive sleep apnoea.

Human schistosomiasis haematobium: effective diagnosis of active infection using a pair of monoclonal antibodies against soluble egg antigen.

The present study was designed to prepare monoclonal antibodies (MAbs) against Schistosoma haematobium soluble egg antigen (SEA) with immunodiagnostic potential for urinary schistosomiasis. From a panel of MAbs, a pair of IgG1 MAbs (2D/11C and 10B/2C) specific for S. haematobium SEA was selected. Both MAbs recognized one band with a 42-kDa molecular weight by western blots. The pair of MAbs was employed in sandwich ELISA for the detection of circulating schistosome antigen (CSA), one as antigen-capturing antibody and the other as peroxidase-conjugated antigen-detecting antibody. The lower detection limit of the assay was 1 ng/ml of S. haematobium SEA. The assay was performed on sera of 65 S. haematobium-infected patients, 25 patients infected with other parasites (Fasciola hepatica, Echinococcus granulosus), and 20 noninfected individuals. CSA was demonstrated in 89% of the S. haematobium-infected group. However, CSA was negative in the sera of healthy individuals and patients infected with other parasites, giving an overall specificity of 100% for the CSA assay. A positive correlation (r=0.37, p<0.01) was detected between the number of S. haematobium eggs excreted in 10 ml urine and the CSA level detected in the sera of S. haematobium-infected patients. Our data show that the use of anti-S. haematobium MAbs for the detection of CSA provides a sensitive and specific method for the immunodiagnosis of active S. haematobium-infected patients. Moreover, CSA assay using this anti-S. haematobium MAb/ELISA system was proven to correlate with intensity of infection and hence morbidity assessment.

A monoclonal antibody against Schistosoma haematobium soluble egg antigen: efficacy for diagnosis and monitoring of cure of S. haematobium infection.

A monoclonal antibody (mAb), 2F/11F, raised against Schistosoma haematobium soluble egg antigen (SEA) was found to be nonreactive with S. mansoni SEA or other parasite antigens (Fasciola hepatica, Echinococcus granulosus). This IgG1 mAb recognized a repetitive epitope on S. haematobium SEA in the molecular-weight regions of 70, 42, and 35 kDa. It was employed as both an antigen-capture and a biotinylated detection antibody in a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of circulating schistosome antigen (CSA) and had a detection limit of <1 ng S. haematobium SEA/ml. CSA levels were measured in serum and urine samples from 116 S. haematobium-infected rural students before therapy and at 4, 8, and 12 weeks after praziquantel treatment. Serum and urine samples from 50 S. mansoni -infected patients, 15 patients harboring other parasites, and 30 noninfected individuals were also assessed. CSA was detected in 90.5% of serum samples and 94% of urine samples from S. haematobium-infected patients. CSA was undetectable in serum from the 15 patients harboring other parasites and in 94% of serum samples and 84% of urine samples from S. mansoni-infected patients. In the S. haematobium-infected group a positive correlation was detected between CSA levels in serum and urine samples and the egg load per 10 ml urine. A significant reduction in CSA levels was detected in serum and urine samples after praziquantel therapy. CSA was undetectable in 87% of serum samples and 81.5% of urine samples from schistosomiasis haematobium patients at 12 weeks post-treatment. These data demonstrate that the use of mAb 2F/11F for detection of CSA provides a sensitive method for the immunodiagnosis and monitoring of cure of schistosomiasis haematobium.

The coagulation profile in hepatosplenic schistosomiasis.

The biological activity of blood coagulation factors II, V, VII, VIII, IX, X, XI and XII, fibrinogen and prekallikrein was assessed in 15 healthy subjects and 60 patients with endemic Egyptian hepatosplenomegaly. The degree of liver disease was graded according to the Child-Pugh classification, the intensity of S. mansoni infection was monitored by determination of circulating schistosome immune complexes (CSIC) level using a monoclonal antibody and hemostasis activation was detected by measurement of hemostatic markers D-dimer and prothrombin fragment 1 + 2 (F1+2). Functional activity of antithrombin III, alpha2-antiplasmin and protein C as well as quantitative determination of plasma concentrations of alpha1-antitrypsin, C1 activator inhibitor and alpha2-macroglobulin were also carried out. The progressive deterioration of liver function which matched the severity of the disease and the intensity of schistosomal infection led to a reduction in anticoagulant proteins (decreases in antithrombin III and protein C) resulting in hypercoagulability and thrombin generation (increased F1+2) subsequently followed by consumption (prolongation of coagulation screening tests, thrombocytopenia, hypofibrinogenemia and decreased factor VIII resulting in hypocoagulability and secondary fibrinolysis (increased D-dimer and decreased alpha2-antiplasmin). A significant decline in fibrinogen and factors VII, XII and prekallikrein was detected in bleeders compared with ascitic patients. The decline in factor XII was closely related to CSIC high titers in all disease groups, but was not correlated to D-dimer or F1+2 concentrations. This suggests that circulating schistosome immune complexes may exert an inhibitory effect on contact factor XII which should be taken into account when considering the reasons for schistosomal coagulopathy and bleeding in hepatosplenic schistosomiasis.

Fibronectin, platelet factor 4 and beta-thromboglobulin in endemic hepatosplenic schistosomiasis: relation to acute hematemesis.

Some platelet alpha-granule contents were assessed in parallel with other markers of hemostatic imbalance in 50 patients with hepatosplenic schistosomiasis (15 patients with compensated hepatosplenomegaly, 15 patients with advanced hepatic fibrosis and ascites and 20 patients during an acute attack of hematemesis from ruptured esophageal varices). Platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), fibronectin (FN), prothrombin fragment 1 + 2, thrombin-antithrombin (TAT) complexes, fibrin degradation products (FbDP) and D-dimer were assessed in schistosomal patients compared to controls (15 healthy subjects). A significant increase in both thrombin (high TAT and prothrombin fragment 1 + 2 levels) and plasmin (high FbDP and D-dimer levels) generation was detected in decompensated patients establishing the presence of a steady state of low-grade disseminated intravascular coagulation, with and without overt bleeding, in these patients. A decrease in plasma FN concentration was found in diseased groups compared to controls. The reduction in plasma levels of FN paralleled the defective liver function and matched the relative decrease in tissue FN in liver specimens of decompensated patients suggesting that FN levels can be used to evaluate the pathological staging of the disease. A significant increase in beta-TG and PF4 levels was noted in decompensated patients with ascites and/or acute hematemesis compared both to controls and compensated patients reflecting platelet alpha-granule release and consequently increased in vivo platelet activation which may initiate and/or perpetuate the pathophysiological mechanisms of the hemostatic imbalance underlying the hemorrhagic diathesis in hepatosplenic schistosomiasis.

Hyperfibrinolysis in hepatosplenic schistosomiasis.

AIM: To evaluate the nature of accelerated fibrinolysis in hepatosplenic schistosomiasis. METHODS: The biological activity of plasminogen (Plg), plasminogen activators (PA), alpha 2-antiplasmin (alpha 2-AP) and plasminogen activator inhibitor-1 (PAI-1) was determined by photometric analysis in 15 compensated and 35 decompensated patients with endemic Egyptian hepatosplenomegaly. Quantitative measurement of plasma concentrations of tissue t-PA, t-PA-PAI-1 complex, alpha 2-antiplasmin-plasmin complex (alpha 2-APP), fibrinogen degradation products (FbDP), D-dimers (D-D), thrombin-antithrombin complex (TAT) and prothrombin fragment (F 1 + 2) complexes, using double antibody sandwich enzyme linked immunosorbent assays and grading of the degree of hepatic insufficiency according to the Child-Pugh classification, were also carried out. RESULTS: The progressive deterioration of liver function in schistosomal patients, which matched the severity of the disease, led to simultaneous defects in profibrinolytic (decreased Plg and increased PA and t-PA) and antifibrinolytic (decreased alpha 2-AP and PAI-1) factors-the latter defects being the most prominent-resulting in significant generation of plasmin (increased APP complexes) and therefore enhanced fibrinolysis (increased FbDP and D-dimer). The raised concentrations of FbDP, D-D, TAT and F 1 + 2 established its secondary nature. CONCLUSION: These findings suggest that the amount of PAI-1 available to bind and neutralise circulating t-PA may be a critical factor in the progress of hyperfibrinolysis observed in hepatosplenic schistosomiasis, and that the pronounced reduction in its plasma concentration may be regarded as a potential warning indicator of haemostatic imbalance in decompensated schistosomal patients at high risk of variceal bleeding.

Isolation and concentration of microfilariae from peripheral blood of Wuchereria bancrofti infected patients by density gradient centrifugation.

A Ficoll-Hypaque gradient centrifugation technique was used for isolation and concentration of microfilariae from peripheral blood of 30 subjects with clinically and parasitologically diagnosed Wuchereria bancrofti infections. 86% of the microfilariae were found in the Ficoll-Hypaque layer. None were detected in the plasma, leucocyte layer or lower erythrocyte layer. 14% of microfilariae were identified on the top part of the erythrocyte layer. A 35 fold concentration and 88% quantitative recovery of parasites was achieved by conventional centrifugation of microfilariae-rich Ficoll-Hypaque layer. Following the centrifugation procedures, living motile microfilariae were separated. These results indicate that Ficoll-Hypaque centrifugation technique could be an effective method for the detection of low levels of microfilaraemia, and for obtaining relatively pure suspensions of living microfilariae for metabolic studies, production of antigen-rich excretory-secretory products and antigen analysis.

Antifilarial IgM versus IgG antibody determination in the diagnosis of Wuchereria bancrofti infection in Egyptians.

Circulating antifilarial IgM and IgG antibodies were assessed by indirect ELISA in 184 serum specimens from 80 patients with clinically and parasitologically diagnosed filarial infections (20 with acute filariasis 40 with chronic filariasis & 20 asymptomatic microfilaraemic subjects), 64 individuals with other parasitic infections, 20 parasitologically-free subjects from filariasis endemic areas and 20 normal healthy controls. A soluble surface membrane extract from Dirofilaria immitis worms was used as the antigen. Using a single serum dilution of 1:128 and optical densities (OD) at 492 nm, the respective cut off values for IgM and IgG were found to be 0.24 and 0.22. All healthy non-endemic controls were seronegative by IgM and IgG ELISAs. The highest antifilarial IgM OD492 values were obtained in 20 patients with acute filariasis (95% sensitivity), while the highest antifilarial IgG OD492 values were observed n 40 patients with chronic filariaisis (97.5% sensitivity). Asymptomatic microfilaraemic subjects gave IgM and IgG OD492 values which were significantly lower than those of other forms of clinical disease and endemic control subjects. The antifilarial IgM and IgG respective sensitivities in asymptomatic subjects were 75% and 70%. Endemic controls had positive antifilarial IgM (65%) and IgG (75%) levels. Of 64 subjects with other parasites only one with Ancylostoma duodenale had positive IgM level (98.4% specificity); while 9 patients with nematodal infections mainly had false positive antifilarial IgG antibody levels (85.9% specificity). These results suggest that measuring circulating antifilarial IgM antibody level may have some diagnostic advantage over measuring IgG antibody level for the detection of active filarial infection and consequently better management of the disease.

Effect of praziquantel on certain immune responses of schistosomal Egyptian patients. II. Cell-mediated lymphoproliferative response.

The lymphoproliferative blastogenic responses to mitogens, PHA and Con A, and to schistosome-derived antigens. S. mansoni worm and egg, were tested in 35 schistosomal patients and 10 healthy controls. Of the former group, 18 patients had intestinal mansoniasis and 17 had mansoniasis with hepatosplenomegaly. The test was repeated 2 weeks and 1 and 2 months after treatment with praziquantel. The delayed intradermal test for schistosomiasis was performed on 25 of the schistosomal patients and was repeated 1 month after treatment. Statistical analysis of results of lymphoproliferative blastogenic responses showed no significant differences between the control and the two schistosomal groups in response to mitogens. The group with intestinal mansoniasis responded significantly to both schistosomal antigens, compared to the control and hepatosplenic groups. Their proliferative responses showed a significant rise 2 weeks after treatment, then a gradual drop at 1 and 2 month intervals. The hepatosplenic group responded significantly to worm antigen before treatment; their proliferative responses to both schistosomal antigens showed a significant rise 2 weeks after treatment and remained raised thereafter. No relationship was established between either of the two schistosomal groups for age, intensity of infection or positive delayed intradermal reaction.

Broad spectrum antifungal agents in otomycosis.

The in vitro inhibitory activities of four currently used antimycotic agents (clotrimazole, econazole, miconazole and cyclopirox-olamine) against 304 fungal isolates comprising 51 species from 14 genera of moulds and yeasts, using a serial dilution procedure, were studied. Clotrimazole and econazole were found to have a grossly similar broad-spectrum antifungal activity, inhibiting all the tested yeasts and moulds at a concentration ranging from 0.1-4 micrograms/ml. At this range miconazole inhibited 90 per cent of the strains and cyclopirox-olamine inhibited 57 per cent only and thus they were less effective. Econazole 1 per cent solution was very effective in vivo in the treatment of otomycosis within 1-3 weeks. The drug was well tolerated, with no side-effects. Owing to the high broad-spectrum antifungal activity of clotrimazole and econazole, they should be the treatment of choice in otomycosis and can be used safely as otic drops.

Accelerated onset of albuminuria in diabetic rats preimmunized with glucosylated rat serum albumin.

Young adult rats were immunized with either in vitro glucosylated or unmodified rat serum albumin (RSA) followed by induction of the diabetic state. At various times after streptozotocin administration, sera were evaluated for specific antibody response using the enzyme-linked immunosorbent assay while urine was monitored for the presence of albumin by radial immunodiffusion. The data indicate that glucosylated RSA is capable of eliciting the production of antibodies, but unmodified RSA is not. The specificity of the generated antibodies appears to be directed toward the glucitol-lysine residues of the modified albumin. Preimmunization of rats with glucosylated RSA followed by induction of diabetes causes a rapid decline in specific antibody titers and greatly enhances the rate of progression to albuminuria. These results demonstrate that in vitro and in vivo glucosylation of an endogenous protein provide products with identical antigenicity. They also suggest an animal model for the study of diabetic autoimmunity involving glucosylated endogenous proteins.

'Candida infection in the tongue and pharynx'.

60 patients with chronic pharyngitis were investigated. In all cases the tongue was coated, fissured or hairy. Scrapings from the tongue and swabs from the posteriors pharyngeal wall were examined for the presence of Candida albicans. In the tongue cultures were positive in all cases, and in the pharynx they were positive in 42 cases. After treatment with 'Canasten' solution, the condition was cured in 53 cases. This directs attention to the role of Candida albicans as a cause of chronic pharyngitis, and to the relationship between candidiasis of the tongue and that of the throat.

Glucosylated collagen is antigenic.

Sera from rats inoculated with in vitro glucosylated rat skin acid-soluble collagen were evaluated for specific antibody response using the enzyme-linked immunosorbent assay. The data indicate that the altered protein is capable of eliciting the production of antibodies, but unmodified collagen is not. The specificity of the generated antibodies appears to be directed toward the glucitollysine residues of the modified collagen. Sera from streptozotocin-induced diabetic rats contain antibodies that clearly bind glucosylated collagen. These results provide support for the hypothesis that in diabetes mellitus increased collagen glucosylation during chronic hyperglycemia may be the initiating event of an autoimmune response leading to the long-term complications.

Cryosurgery in cutaneous leishmaniasis.

Thirty patients with cutaneous leishmaniasis were treated with cryotherapy using a CO2 cryomachine and all were cured without noticeable scarring within 4-5 weeks, with no relapse. Histopathological examination showed that cryotherapy eradicated all parasites in less than 1 hour. Leishmania tropica, L. ethiopica and L. brasiliensis are all markedly thermosensitive and thus cryotherapy seems parasiticidal to all types. Adequate cryotreatment of cutaneous leishmaniasis will preclude the development of mucocutaneous extension, and its use in mucocutaneous disease is also recommended.

Maxillary sinus hypoplasia and superior orbital fissure asymmetry.

Misdiagnosis of maxillary sinus hypophasia usually as sinus infection, sometimes as neoplasm, can lead to unnecessary and difficult surgical explorations. Associated anatomical abnormalities, e.g., caudal displacement of the ipsilateral frontal lobe of the brain or central position in the maxilla of the infraorbital nerve may create unexpected surgical hazards. Associated orbital enlargement can lead to diagnostic confusion in the investigation of headache, especially if the superior orbital fissures show marked asymmetry suggesting erosion. Projection of the fissure into the antrum in Waters view can simulate trabeculation of the sinus or fracture of the inferior orbital rim. Radiologic examination of 500 patients without intracranial or intraorbital lesions revealed maxillary sinus hypoplasia in 36 cases (7.2%) and in half the hypoplasia was unilateral (sinus asymmetry); aplasia was not encountered. Fissure asymmetry was present in 30 cases (6%), being present in 3.66% of patients with normally developed sinuses and in 36.1% of the hypoplasia patients. The appearances and measurements of the fissure are presented and examples of marked normal fissure asymmetry are demonstrated. Maxillary sinus hypoplasia is classified in this series as grade I-mild hypoplasia with limited inferolateral expansion (4 cases) and grade II in which there is also a curved orbital floor and lateral displacement of the adjacent nasal wall (32 cases).

Non-invasive antromycosis: (diagnosis and treatment).

Increasing numbers of cases of chronic maxillary sinusitis are encountered which resist frequent sinus irrigation and treatment of predisposing factors. The antral washouts are of unusual consistencies and colours. The antral washouts of six cases were investigated by culture and microscopic examination and proved to contain fungal colonies. Antroscopy was a valuable asset in diagnosis. One patient had a Caldwell-Luc operation and daily irrigation through an indwelling polythene tube followed by daily instillation of clotrimazole (Canesten). The five patients who refused operation were treated by repeated bi-weekly antral washouts followed by instillation of clotrimazole. Weekly samples of antral secretions were examined by culture and microscopic examination until they were free for four consecutive weeks. We believe that frequent antral irrigation and local instillation of a broad-spectrum antimycotic drug--preferably after debridement--is the treatment of choice for antromycosis.