James A. Koziol

Matrix Metalloproteinases Increase Very Early during Experimental Focal Cerebral Ischemia

Microvascular integrity is lost during focal cerebral ischemia. The degradation of the basal lamina and extracellular matrix are, in part, responsible for the loss of vascular integrity. Matrix metalloproteinases (MMPs) may play a primary role in basal lamina degradation. By using a sensitive modification of gelatin zymography, the authors investigated the activity of MMP-2 and MMP-9 in frozen 10-µm sections of ischemic and nonischemic basal ganglia and plasma samples of 27 non-human primates after middle cerebral artery occlusion/reperfusion (MCAO/R) for various periods. The gelatinolytic activities were compared with parallel cell dUTP incorporation in the ischemic zones of adjacent sections. In the brain, the integrated density of MMP-2 increased significantly by 1 hour after MCAO and was persistently elevated thereafter. Matrix metalloproteinase-2 expression was highly correlated with the extent of neuron injury and the number of injured neurons (r = 0.9763, SE = 0.004, 2P < 0.0008). Matrix metalloproteinase-9 expression only was significantly increased in subjects with hemorrhagic transformation. In plasma, only MMP-9 increased transiently at 2 hours of MCAO. These findings highlight the early potential role of MMP-2 in the degradation of basal lamina leading to neuronal injury, and an association of MMP-9 with hemorrhagic transformation after focal cerebral ischemia.

The Natural History of Interstitial Cystitis: A Survey of 374 Patients

A survey directed at determining the natural history of interstitial cystitis was conducted at our clinic. Information on demographics, risk factors, symptoms, pain and psychosocial factors was elicited from 374 patients who satisfied the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases criteria for interstitial cystitis and had all been diagnosed as having interstitial cystitis by a urologist. With regard to demographics, patients were predominantly female (89.8%) and white (94.1%), with a mean age of 53.8 +/- 0.7 years (standard error) and age at the first symptoms of 42.5 +/- 0.8 years. Information on 25 potential risk factors included 44.4% of the women reporting hysterectomy, 38.2% of the patients having strong sensitivities or allergic reactions to medication and only 2.7% being diabetic. With regard to interstitial cystitis symptoms, frequency and urgency were reported by 91.7% and 89.3% of the patients, respectively, while pelvic pain, pelvic pressure and bladder spasms were reported by more than 60% of respondents and burning by 56%. Location and degree of pain were also reported. Urination relieved or lessened interstitial cystitis pain for 73.6% of the patients and medication was effective for 46.8%. Other behaviors (for example hot baths, heating pads, lying down or sitting) were less effective. Conversely, stress, constrictive clothing and intercourse increased interstitial cystitis pain in more than 50% of the patients. In addition, acidic, alcoholic or carbonated beverages, and coffee or tea increased interstitial cystitis pain in more than 50% of the patients. More than 60% of the patients were unable to enjoy usual activities or were excessively fatigued and 53.7% reported depression. Travel, employment, leisure activities and sleeping were adversely affected in more than 80% of the patients. Pain location and degree differed significantly between patients with and without ulcers in the bladder. In addition, there was an apparent plateau in the frequency and urgency among patients after approximately 5 years with symptoms.

Cladribine in treatment of chronic progressive multiple sclerosis

Chronic progressive multiple sclerosis (MS) is a severely disabling demyelinating disease in which autoimmune processes seem to have a major role. The nucleoside drug cladribine is a potent lympholytic agent with few side-effects. We have studied its efficacy and safety in a randomised double-blind trial. 51 patients (48 entered as matched pairs) received four monthly courses of 0.7 mg/kg cladribine or placebo (saline) given through a surgically implanted central line. Neurologists with no knowledge of which medication the patient was receiving examined the patients monthly and noted two rating scale scores (Kurtzke and Scripps). Cerebrospinal fluid and brain magnetic resonance imaging (MRI) examinations were done at 6 and 12 months. Average neurological scores, demyelinated volumes on MRI, and concentrations of oligoclonal bands in cerebrospinal fluid were stable or improved in the patients receiving cladrabine but continued to deteriorate in patients on placebo. Mean paired (placebo minus matched cladribine) differences at 12 months relative to baseline were 1.0 (SE 0.4) for the Kurtzke scores, -13.9 (2.3) for the Scripps scores, 4.57 (1.17) mL for demyelinated volumes, and 7.3 (3.3) arbitrary units for concentrations of oligoclonal bands. Cladribine was generally well tolerated and clinically significant toxicity occurred in only 1 patient, in whom severe marrow suppression developed with complete recovery after several months. 1 patient died of newly acquired hepatitis B, an event unlikely to be related to cladribine. We conclude that the immunosuppressive drug cladribine influences favourably the course of chronic progressive MS.

Significance of regional wall thickening abnormalities relative to transmural myocardial perfusion in anesthetized dogs.

In 15 open-chest, anesthetized dogs, regional systolic wall thickening (%AWT) was measured with sonomicrometry and regional blood flow was determined with tracer microspheres (7–10, m) before and after various degrees of coronary artery narrowing were created with a hydraulic occluder. The stenoses were categorized into four groups by the effect on %AWT, and the corresponding myocardial blood flow (MBF) was determined in four layers across the ventricular wall (layer 1: subendocardium; layer 4: subepicardium). In group 1, %AWT decreased 44 ± 10% and only layer 1 MBF was significantly reduced (-28%); in group 2, %AWT decreased 77 8% and MBF was reduced in both layers 1 and 2 (-52% and -36%, respectively); in group 3, %AWT decreased 104 i 3% and MBF was reduced in the three inner layers (layer 1: -65%; layer 2: -58%; layer 3: -34%); in group 4, %AWT decreased 145 ± 9% (systolic wall thinning) and transmural reduction of MBF was found (layer 1: -74%; layer 2: -68%; layer 3: -55%; layer 4: -29%). We conclude that (1) up to 75% reduction in systolic wall thickening may occur when perfusion to only the inner one-half of the myocardium is decreased; (2) akinetic wall motion may be observed when perfusion remains normal in the subepicardial one-fourth of the wall; and (3) dyskinesia (wall thinning) occurs when blood flow is reduced transmurally.

Focal cerebral ischemia induces active proteases that degrade microvascular matrix

Background and Purpose— Focal cerebral ischemia causes microvessel matrix degradation and generates proteases known to degrade this matrix. However, proof that the proteases generated do indeed degrade vascular matrix is lacking. Here we demonstrate that active proteases derived from ischemic tissue after middle cerebral artery occlusion (MCAO) and transferred to normal tissue can degrade vascular matrix. Methods— In an ex vivo bioassay, the effects of supernatants from ischemic and normal basal ganglia of nonhuman primates, proteases, and control buffer on the immunoreactivity of vascular matrix constituents in normal brain tissue sections were quantified. Protease families were identified with specific inhibitors. Results— Plasmin, active matrix metalloproteinase (MMP)-2, and active MMP-9 significantly reduced microvessel-associated collagen, laminin, and heparan sulfate proteoglycans (HSPG). The vascular HSPG perlecan was more sensitive than collagen or laminin in the bioassay and in the ischemic core 2 hours after MCAO. Two-hour and 7-day ischemic tissue samples significantly degraded matrix perlecan and collagen. Inhibitor studies confirmed that while active MMPs were generated, active cysteine proteases significantly degraded microvessel perlecan. The cysteine proteases cathepsins B and L were generated in the microvasculature and adjacent neurons or glial cells 2 hours after MCAO and decreased perlecan in the bioassay. Conclusions— This is the first direct evidence that active proteases are generated in ischemic cerebral tissues that are acutely responsible for vascular matrix degradation. Degradation of vascular perlecan, the most sensitive matrix component thus far identified, may be due to cathepsins B and L, generated very rapidly after MCAO.

Extended Follow-Up of Patients With Hairy Cell Leukemia After Treatment With Cladribine

PURPOSE Hairy cell leukemia (HCL) is an uncommon, indolent, chronic B-cell lymphoproliferative disorder involving the marrow and spleen. Therapy for HCL includes splenectomy, interferon alfa-2a and alfa-2b, pentostatin, and cladribine. The purpose of this article was to report the extended follow-up of HCL patients treated with cladribine. PATIENTS AND METHODS Two hundred nine patients with HCL who were treated with cladribine had at least 7 years of follow-up. A course of cladribine constituted a 7-day continuous intravenous infusion at a dose of 0.1 mg/kg/d. RESULTS Of the 207 assessable patients who had at least 7 years of follow-up, 196 (95%) achieved a complete response (CR) and 11 (5%) achieved a partial response (PR) after a single course of cladribine (overall response rate, 100%). The median first-response duration for all responders was 98 months. Seventy-six patients (37%) experienced relapse after their first course of cladribine. The median time to first relapse for all responders was 42 months. Time to treatment failure of CRs compared with PRs was statistically significant (P <.0005). The overall survival rate was 97% recorded at 108 months. Forty-seven patients developed 58 second malignancies. The observed-to-expected ratio for second malignancies was 2.03 (95% confidence interval, 1.49 to 2.71). CONCLUSION These results confirm previous observations that single courses of cladribine administered to patients with HCL induce high response rates, the majority of which are CRs. Most patients enjoy long-lasting complete remissions, and those patients who experience relapse can be successfully re-treated with cladribine.

Profiles of Matrix Metalloproteinases, Their Inhibitors, and Laminin in Stroke Patients Influence of Different Therapies

BACKGROUND AND PURPOSE The goal of this study was to determine the temporal profile of several matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), and laminin (an MMP substrate) in human stroke under different treatment paradigms, including thrombolysis and hypothermia. METHODS We serially measured the serum levels of MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, and laminin in 50 patients with acute ischemic stroke using zymography or enzyme-linked immunosorbent assay. Patients were treated with heparin, therapeutic thrombolysis, or hypothermia. Scandinavian Stroke Scale scores were obtained at baseline. Infarct volume was measured with CT scanning on day 4 after stroke onset. Healthy persons were used as control subjects. RESULTS MMP-2 and MMP-9 increased during the course of ischemia, whereas intact laminin and TIMP-2 decreased significantly (P<0.05). MMP-9 and laminin levels varied significantly by infarct size (P=0.001) and therapy (P=0.0005). MMP-9 levels were significantly higher in patients treated with tissue plasminogen activator (tPA) compared with patients treated with hypothermia. The cleaved form of MMP-9 was found solely in 4 patients treated with tPA. Intact laminin levels were significantly lower in the tPA group than in the hypothermia group. CONCLUSIONS Selected MMPs and TIMPs are involved in the pathophysiology of acute stroke. This is also reflected by changes in laminin. Treatment paradigms differentially influence levels of MMP-9 and laminin. Combination therapies explicitly involving MMP inhibition could be of value in future treatment strategies.

Fibrin contributes to microvascular obstructions and parenchymal changes during early focal cerebral ischemia and reperfusion.

Ischemic cerebral injury is associated with activation of the blood coagulation cascade. To elucidate the contribution of fibrin formation to microvascular injury during focal cerebral ischemia and reperfusion, we have studied the time course and the localization of fibrin deposition in cerebral microvessels and the surrounding tissues during ischemia/reperfusion in a well-described nonhuman primate model. Methods Cerebral tissues from adolescent male baboons were examined after 2-hour middle cerebral artery occlusion (n=3) and after 3 hours of middle cerebral artery occlusion and 1-hour (n=6), 4-hour (n=3), and 24-hour (n=4) reperfusion; tissues from control primates (n=3) also were examined. Fibrin deposition was detected by immunohistochemical techniques using the fibrin-specific monoclonal antibody MH-1. The number and size distribution of microvessels associated with fibrin were quantified by video-imaging microscopy. Results Fibrin was associated with microvessels only in the ischemic zone where severe neuronal injury was documented, its frequency increasing with the reperfusion period (F4.26=3.80, P<.05). Extravascular fibrin deposition was significantly increased by 24-hour reperfusion compared with the other subjects (P<.05). Preischemia infusion of the anti-tissue factor monoclonal antibody TF9-6B4 resulted in significant reduction of intramicrovascular fibrin (P<.038 versus no intervention) at 1-hour reperfusion but had no effect on extravascular fibrin deposition. Conclusions These results suggest that microvascular fibrin deposition accumulates in a time-dependent manner during focal cerebral ischemia/reperfusion and that exposure of plasma to perivascular tissue factor is partially responsible for occlusion formation. During ischemia the large plasma protein fibrinogen extravasates and interacts with parenchymal tissue factor, forming significant extravascular fibrin by 24 hours of reperfusion.

Human recombinant erythropoietin promotes differentiation of murine megakaryocytes in vitro.

To determine if erythropoietin affects megakaryocytopoiesis, we measured acetylcholinesterase (AchE) activity, a marker of the murine megakaryocytic lineage, after the addition of human recombinant erythropoietin to serumless murine bone marrow cultures. Erythropoietin increased AchE activity substantially. Moreover, when the hormone was added to serumless cultures of 426 isolated single megakaryocytes derived from megakaryocytic colonies, erythropoietin induced a significant increase in the diameters of these cells. From a Bayesian analysis of the likelihood that some megakaryocytes increased in DNA content during the culture period, we estimate that 61% of the cells increased in ploidy. These data indicate that the action of erythropoietin is not restricted to the erythroid lineage.

Rapid Disruption of an Astrocyte Interaction With the Extracellular Matrix Mediated by Integrin α6β4 During Focal Cerebral Ischemia/Reperfusion

Background and Purpose Integrins participate in cerebral microvascular integrity and signaling during focal ischemia/reperfusion. The integrin subunits α1, α6, and β1 are distributed identically on normal cerebral microvessels. Studies in epithelium indicate that integrin α6β4, which interacts with laminin-5 in the basal lamina/extracellular matrix, is unique. This study describes the exact location of α6, β4, and α6β4 and that their responses in focal cerebral ischemia are relevant to astrocyte-matrix interactions. Methods The effect of middle cerebral artery occlusion and subsequent reperfusion on the microvascular expression of α6β4 and laminin-5 in regions of cellular injury (dUTP incorporation) was examined in 15 nonhuman primates. Well-characterized antibodies against human α6, β4, α6β4, laminin-5 and laminin-1, endothelial CD31, and vascular markers were measured with computerized video imaging and laser confocal microscopy. Results Integrin α6β4 was localized on astrocytes where it connects with the extracellular matrix at the astrocyte-vessel interface. It represented 59.3±16.4% of α6 antigen in cerebral microvessels <100 μm in diameter. By 2 hours of ischemia, the significant reduction in α6 expression (2 P <.001) was accompanied by decreases in β4/laminin-5 (0.76±0.03 to 0.20±0.09; 2 P =.001) and α6β4/laminin-5 (0.73±0.18 to 0.25±0.11; 2 P =.001) in the region of dUTP incorporation. Parallel changes in laminin-5 and laminin-1 were less pronounced and coincided by 24 hours. Conclusions This is the first description of a potential role of integrin α6β4 in the brain, where it mediates astrocyte-matrix interactions. The dramatic disappearance of α6β4 relative to its ligands reflects early loss of integrity between the astrocyte and the vessel wall in selected microvessels in response to ischemia.